Compositions and methods for rescuing retinal and choroidal structure and function

Abstract

The present invention relates to the use of anti-hemichannel compounds, including anti-connexin 43 hemichannel opening compounds, in rescuing or restoring retinal function, rescuing or restoring retinal structure, and / or rescuing or restoring choroidal structure and / or function in chronic retinal disorders and other disorders.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 900,379, filed September 13, 2019, and U.S. Provisional Patent Application No. 62 / 903,504, filed September 20, 2019, both of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention generally relates to the retina and choroid, as well as other ocular processes, and to connective protein hemichannels.

[0004] By incorporating via reference

[0005] All U.S. patents, U.S. patent application publications, foreign patents, foreign and PCT publications, articles and other documents, references and publications cited herein, and all references listed as cited in any patent herein, are hereby incorporated in their entirety by reference. The information contained herein is also part of this application, as all text and other content are repeated in the application, and will be considered part of the text and content of this application. Background Technology

[0006] The following contains information that may be useful for understanding the invention. No information, publication, or document specifically or implicitly referenced herein is acknowledged to be prior art or essential to the invention described or claimed herein.

[0007] Diabetes is an increasingly common condition in which the body develops resistance to the hormone insulin. This prevents sugar, or glucose, from leaving the bloodstream and entering cells. This condition can lead to serious complications, including an eye-related disease called diabetic retinopathy.

[0008] When a person is in a state of hyperglycemia for an extended period, the walls of small blood vessels throughout the body thicken. This makes it more difficult for oxygen and essential nutrients to be transported from the blood to cells that depend on these nutrients and oxygen for survival. Areas significantly affected include the retina at the back of the eye. Poor circulation in these tiny blood vessels can also lead to leakage. When blood leaks out from them, it can get stuck in the retina, reducing its ability to convert light waves into vision. Furthermore, choroidal thickness is altered in diabetic patients and may be associated with the severity of retinopathy. The presence of diabetic macular edema is associated with a significant reduction in choroidal thickness. See Regatieri CV, Branchini L, Carmody J, Fujimoto JG, Duker JS, Choroidal thickness in patients with diabetic retinopathy analyzed by spectral-domain optical coherence tomography. Retina. Mar 2012; 32(3): 563-8.

[0009] As blood vessels are damaged by diabetes, the overall condition of the retina deteriorates rapidly. Leaking blood can block the retina, and the reduction in nutrients and oxygen can lead to tissue death. Without treatment, the end result is vision loss, eventually leading to complete loss of sight.

[0010] It is estimated that most people will develop some signs of mild diabetic retinopathy twenty years after being diagnosed with diabetes. The pathological process of diabetic retinopathy involves microaneurysms and punctate hemorrhages in the retina. Tiny swollen blood vessels and / or hemorrhages under the choroid can damage receptor cells and retinal neurons and can lead to blindness.

[0011] According to the National Eye Institute (NEI), diabetic retinopathy typically progresses through a series of four stages: (1) Mild nonproliferative retinopathy: This stage involves small areas of swelling in the retinal vessels, called microaneurysms. (2) Moderate nonproliferative retinopathy: As the disease progresses, ophthalmologists may now be able to see significant swelling and deformation of the retinal vessels. At this stage, they may also lose their ability to transport oxygen and nutrients. (3) Severe nonproliferative retinopathy: This stage sees worsening of vascular occlusion, with parts of the retina losing blood. New vessels may also grow, blocking areas of the retina. (4) Proliferative diabetic retinopathy (PDR): Finally, these newly grown vessels proliferate within the retina, leading to leakage, vision loss, and scar tissue, which can result in retinal detachment and blindness.

[0012] There are two main treatment options for diabetic retinopathy: injections and laser surgery. Injections involve injecting medications, such as corticosteroids or vascular endothelial growth factor (VEGF) antagonists, directly into the eye. In surgery, doctors can use a laser to burn parts of the retina. By effectively killing these areas, the limited available blood supply can flow to the remaining living tissue, thus helping to protect vision.

[0013] Unfortunately, there is no known cure for diabetic retinopathy. The damage caused by blood vessel growth, leakage, and lack of oxygen is permanent, and diabetic retinopathy is not entirely reversible with current treatments.

[0014] Although the choroid is a key part of the metabolic transport system to the outer layers of the retina, little is known about it. (Zouache and Luthert, The Choroid In AMD: A Critical Point of Failure? RetinaSpecialist, January 8, 2018). As one of the two main blood supplies to the retina, the choroid supplies blood to the outer RPE, photoreceptors, and some overlying tissue layers. Choroidal failure plays a role in the pathogenesis of age-related macular degeneration. Changes in the choroid have been reported in both early and late AMD. Furthermore, the vascular density of the choroidal capillary layer is significantly lower than in normal macula in macula exhibiting basal lamellar deposition, geographic atrophy, and discoid scarring. (Zouache and Luthert, ibid.). Importantly, patients with choroidal changes are at risk of developing retinal vein occlusion. Treatment for abnormal choroidal structure and function is needed.

[0015] This patent relates to the important discovery of methods and compositions comprising anti-half-channel compounds that can fundamentally reverse diabetic retinopathy and restore retinal and choroidal structure and function in this disease and other diseases, disorders and conditions. Summary of the Invention

[0016] The invention described and claimed herein has many attributes and embodiments, including, but not limited to, those set forth, described, or mentioned in this brief overview. It is not intended to be all-encompassing, and the invention described and claimed herein is not limited to or restricted by the features or embodiments identified in this introduction, but is included for illustrative purposes only and not for limitation.

[0017] This patent relates to methods and compositions for restoring and salvaging retinal structure and function using anti-half-channel compounds. Even single doses have been found to be useful over extended periods. This patent also relates to methods and compositions for restoring and salvaging choroidal structure and function using anti-half-channel compounds.

[0018] For example, data show that anti-hemiplet compounds can be used to enhance and restore retinal function, including in chronic retinal diseases, conditions, and disorders. Among other things, data show that anti-hemiplet compounds can improve the function of photoreceptors and bipolar cells in the inner retina. For example, they also indicate that anti-hemiplet compounds can protect, enhance, and restore inner retinal cells and improve inner retinal function, improve and restore phototransduction pathways and post-photoreceptor neuronal responses, and improve and restore retinal layer structure. Anti-hemiplet compounds were also found to preserve and enhance retinal layer structure (as measured by OCT), and choroidal structure was also improved and restored.

[0019] This patent also relates to methods and compositions for reversing chronic eye diseases previously considered intractable using anti-semichannel compounds. This patent describes the application of anti-semichannel compounds to not only protect and improve, but also salvage and restore retinal function in chronic eye diseases, disorders, and conditions where retinal and / or choroidal damage was previously considered substantially irreversible, including, for example, diabetic retinopathy, nonproliferative diabetic retinopathy (NEI stages 1, 2, and / or 3, designated as “mild,” “moderate,” and “severe” nonproliferative retinopathy), diabetic macular edema, inflammatory or infectious choroiditis, uveitis, age-related macular degeneration (wet and dry), geographic atrophy, and other chronic retinal disorders characterized wholly or partially by loss of retinal structure and / or function.

[0020] This patent also describes the use of anti-semichannel compounds to treat choroidal disorders characterized by complete or partial loss of choroidal structure and / or function. The methods, compounds, and compositions of this invention can be used not only to protect and improve but also to salvage and restore choroidal structure and / or function.

[0021] This patent also describes the use of orally delivered anti-half-channel compounds for restoring retinal function in patients with the disease, and the use of orally delivered anti-half-channel compounds for reversing or substantially reversing chronic retinal diseases.

[0022] This patent also describes the use of orally delivered anti-hemispheric compounds to salvage retinal function in patients with chronic eye diseases. This patent also describes the use of orally delivered anti-hemispheric compounds to salvage retinal structure in patients with chronic eye diseases.

[0023] This patent also describes the use of orally delivered anti-semichannel compounds to salvage the choroidal structure and function in patients in need.

[0024] In another aspect, this patent also relates to the use of anti-half-channel compounds to protect against diabetic retinopathy secondary to spontaneous and chronic systemic hyperglycemia, and to reverse any existing diabetic retinopathy.

[0025] This patent also relates to methods of using anti-half-channel compounds for these purposes, including, for example, tonabosar, a benzopyran compound (cis-6-acetyl-4S-(3-chloro-4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3S-ol (SB-220453, also known as Xiflam or tonabosar).

[0026] For example, in one aspect, the present invention relates to the use of anti-half-channel compounds to reverse retinal and choroidal damage in subjects suffering from diabetes or other conditions characterized entirely or partially by loss of retinal and / or choroidal structure and / or function.

[0027] This patent describes, in one aspect, the use of compounds and methods to modulate connexin hemichannels, including the connexin 43 hemichannel, to salvage or restore retinal function. It also describes the use of compounds and methods to modulate connexin hemichannels, including the connexin 43 hemichannel, to salvage or restore retinal structure.

[0028] This patent describes, in one aspect, the use of compounds and methods to modulate connexin hemichannels, including the connexin 43 hemichannel, to rescue or restore choroidal function. It also describes the use of compounds and methods to modulate connexin hemichannels, including the connexin 43 hemichannel, to rescue or restore choroidal structure.

[0029] On the other hand, by way of example, it also describes the use of anti-half-channel compounds, including anti-connectin 43 half-channel opening compounds, to maintain choroidal structure and function, maintain retinal structure and function, restore retinal function, salvage retinal function, and protect against and reverse diabetic retinopathy secondary to spontaneous and chronic systemic hyperglycemia.

[0030] The methods of the present invention can be used to salvage and restore choroidal structure and function in subjects, restore retinal function, rescue retinal function, and protect against and reverse diabetic retinopathy and macular edema secondary to spontaneous and chronic systemic hyperglycemia, by administering anti-half-channel compounds to subjects who will benefit therefrom, as well as for other chronic retinal disorders mentioned herein.

[0031] Another object of the present invention is to provide compounds, compositions, formulations, kits, dosages, and methods for treating diseases, disorders, and conditions that will benefit from the restoration or salvage of retinal structure, the salvage of retinal function, and / or the restoration of retinal function.

[0032] Another object of the present invention is to provide compounds, compositions, formulations, kits, dosages, and methods for treating diseases, disorders, and conditions that will benefit from the restoration or rescue of choroidal structure, the rescue of choroidal function, and / or the restoration of choroidal function.

[0033] Another object of the present invention is to provide compounds, compositions, formulations, kits and methods for treating diseases, disorders and conditions that will benefit from protection against loss of retinal function.

[0034] Another object of the present invention is to provide compounds, compositions, formulations, kits and methods for treating diseases, disorders and conditions that will benefit from protection against loss of choroidal function.

[0035] In some respects, the treatment methods are applied to mammals, such as humans.

[0036] Anti-half-channel compounds that can be used in this invention include compounds of Formula I, such as Xiflam (tornabenza), and / or prodrugs of any of the aforementioned compounds, as well as other anti-half-channel compounds described or incorporated herein by reference. In some embodiments, the half-channel blocker is a small molecule other than Xiflam (tornabenza), for example, the half-channel blocker described in Formula I or Formula II in U.S. Patent Application Publication No. 20160177298 (filed attributable to Colin Green et al., the disclosure of which is hereby incorporated in its entirety by reference).

[0037] Various preferred embodiments include using orally available small-molecule anti-half-channel compounds to treat diseases, disorders, and conditions characterized at least partially by loss of retinal and / or choroidal structure or function, or to treat subjects at risk of or potentially at risk of loss of retinal and / or choroidal structure or function. In one embodiment, treatment with said anti-half-channel compounds (including orally available anti-half-channel compounds) substantially or completely restores retinal and / or choroidal structure or function.

[0038] Other preferred embodiments include using orally available small molecule anti-half-channel compounds to treat subjects who are at risk of or may be at risk of loss of retinal and / or choroidal structure or function.

[0039] Other aspects of the invention include methods for improving or restoring choroidal blood flow in subjects suffering from chronic retinal disorders, comprising administering an effective amount of a semi-channel blocker to the subject.

[0040] Other aspects of the invention include a method for improving or restoring choroidal blood flow to the outer retina in a subject suffering from chronic retinal impairment, comprising administering an effective amount of a semi-channel blocker to the subject.

[0041] Methods for increasing survival and salvaging or restoring retinal and / or choroidal function in subjects with this need include, for example, administering to the subject a survival-promoting dose of N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrotryptene-4-yl]-3-chloro-4-fluorobenzamide (Xiflam). In some embodiments, the survival-promoting dose is about 10 to about 200 mg daily. In other embodiments, the survival-promoting dose is about 20 to about 100 mg daily. These doses can be administered as a single dose or in divided doses, for example, twice daily. Other daily doses, as well as particularly useful weekly, monthly, and implant administration and dosing regimens, have also been discovered and are provided herein.

[0042] In some approaches, increasing survival, salvage, or restoration can treat chronic retinal disorders. In others, chronic retinal disorders include diabetic retinopathy or diabetic macular edema. In still others, survival-enhancing methods treat chronic retinal disorders selected from wet age-related macular degeneration, dry age-related macular degeneration, geographic atrophy, and hypertensive retinopathy.

[0043] In other respects, ways to increase survival, salvage, or recovery from chronic retinal disorders include retinal degeneration, edema, diabetes, ischemic retinal degeneration, retinal vascular occlusion, and central retinal vein occlusion.

[0044] In other aspects of the method of the present invention, the mixed a-wave function and / or the improved mixed b-wave function are improved or normalized.

[0045] In other aspects of the method of the present invention, the function of retinal PII and PIII rods and cones is improved.

[0046] In other aspects of the method of the present invention, retinal ERG function is improved or normalized.

[0047] In other aspects of the method of the present invention, the function of the inner retina is improved or normalized.

[0048] In other aspects of the method of the present invention, photoreceptor function is improved or normalized.

[0049] It also includes methods for increasing survival, salvaging, or restoring retinal structures in subjects with this need, comprising administering to the subject 10 to 200 mg daily of N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrotryptene-4-yl]-3-chloro-4-fluorobenzamide (Xiflam), or 1.4 mg / kg daily, or other doses described herein. In some embodiments, the retinal structures comprise retinal pigment epithelium, retinal vascular endothelium, and / or retinal layer structures. In other embodiments, microaneurysms and / or large aneurysms in the retina are reduced.

[0050] This also includes methods for increasing survival, salvaging, or restoring choroidal function in subjects with this need, including administering 10 to 200 mg daily of N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrotryptene-4-yl]-3-chloro-4-fluorobenzamide (Xiflam), or other doses described above or herein. In some embodiments of these methods, choroidal blood flow is improved or normalized. In other embodiments, choroidal blood flow to the outer retina is improved or normalized. In other embodiments of these methods, regulation of choroidal blood flow is improved or normalized.

[0051] This article also describes methods for increasing the survival of choroidal structures in subjects with this need, including administering 10 to 200 mg daily of N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrotryptene-4-yl]-3-chloro-4-fluorobenzamide (Xiflam) to the subjects. In some embodiments of these methods, choroidal thickness is improved. In other embodiments, the choroidal vascular bed is improved or normalized.

[0052] In some embodiments of the present invention, increasing the survival of retinal function means restoring or salvaging retinal function.

[0053] In other embodiments of the invention, increasing the survival of retinal structures means restoring or salvaging retinal structures.

[0054] In other embodiments of the invention, increasing the survival of choroidal function means restoring or salvaging choroidal function.

[0055] In other embodiments of the invention, increasing the survival of the choroidal structure means restoring or salvaging the choroidal structure.

[0056] In different implementations, the small molecule that blocks, improves, or inhibits the opening of the half-channel is a prodrug of Xiflam (Tonaboza) or an analogue thereof.

[0057] In another aspect, the present invention provides the use of a hemichannel blocker in the preparation of a medicament for treating a subject or disease, disorder, or condition described or mentioned herein. The medicament comprises, is substantially composed of, or is composed of an anti-hemichannel compound. In one embodiment, the anti-hemichannel compound is a small molecule anti-hemichannel compound. In another embodiment, the small molecule anti-hemichannel compound is an orally available small molecule anti-hemichannel compound.

[0058] In one embodiment, the medicament comprises, substantially constitutes, or is composed of a small molecule half-channel blocker (an example of an anti-half-channel compound). In one embodiment, the medicament comprises, substantially constitutes, or is composed of a compound according to Formula I or Formula II in U.S. Patent Application Publication No. 20160177298. In one embodiment, the medicament comprises, substantially constitutes, or is composed of Xiflam (Tonaboza).

[0059] Formula I:

[0060]

[0061] Where Y is C-R1;

[0062] R1 is an acetyl group;

[0063] R2 is hydrogen, C 3-8 Cycloalkyl groups, C-type carbons optionally interrupted by oxygen or substituted with hydroxyl groups 1-6 Alkyl, C 1-6 alkoxy or substituted amino carbonyl, C 1-6 alkyl carbonyl, C 1-6 alkoxycarbonyl, C 1-6 Alkyl carbonyloxy, C 1-6 Alkyl, nitro, cyano, halogen, trifluoromethyl, or CF3S; or the group CF3-A-, where A is -CF2-.

[0064] -CO-, -CH2-, CH(OH), SO2, SO, CH2-O or CONH; or the group CF2H-A′, where A′ is oxygen, sulfur, SO, SO2, CF2 or CFH; trifluoromethoxy, C 1-6 Alkyl sulfinyl, perfluorinated C 2-6 alkylsulfonyl, C 1-6 alkylsulfonyl, C 1-6 Alkoxysulfinyl, C 1-6 Alkoxysulfonyl, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, phosphonyl, arylcarbonyloxy, heteroarylcarbonyloxy, arylsulfinyl, heteroarylsulfinyl, arylsulfonyl or heteroarylsulfonyl, wherein any aromatic moiety is optionally substituted, C1-6 Alkyl carbonyl amino, C 1-6 alkoxycarbonylamino, C 1-6 Alkyl-thiocarbonyl, C 1-6 alkoxy-thiocarbonyl, C 1-6 alkyl-thiocarbonyloxy, 1-mercapto-C 2-7 alkyl, formyl, or aminosulfinyl, aminosulfonyl, or aminocarbonyl, wherein any amino moiety is optionally surrounded by one or two C2 groups. 1-6 Alkyl substitution, or C 1-6 Alkylsulfinylamino, C 1-6 Alkylsulfonylamino, C 1-6 alkoxysulfinylamino or C 1-6 alkoxysulfonylamino, or terminally C 1-6 Alkyl carbonyl, nitro, or cyano-substituted vinyl groups, or -C(C 1-6 alkyl)NOH or -C(C 1-6 Alkyl)NNH2; or optionally with one or two C 1-6 Alkyl or C 2-7 An alkyl-substituted amino group; one of R3 and R4 is hydrogen or C. 1-4 Alkyl and the other is C 1-4 Alkyl, CF3 or CH2X a It contains fluorine, chlorine, bromine, iodine, and C. 1-4 Alkoxy, hydroxy, C 1-4 Alkyl carbonyloxy, -SC 1-4 Alkyl, nitro, optionally marked with one or two Cs 1-4 Alkyl-substituted amino, cyano or C 1-4 alkoxycarbonyl; or R3 and R4 together are optionally C 1-4 Alkyl-substituted C 2-5 Polymethylene;

[0065] R5 is C 1-6 Alkyl carbonyloxy, benzoyloxy, ONO2, benzyloxy, phenoxy or C 1-6 Alkyl groups and R6 and R9 are hydrogen, or R5 is a hydroxyl group and R6 is hydrogen or C 1-2 Alkyl group and R9 is hydrogen;

[0066] R7 is a heteroaryl or phenyl group, both of which are independently and optionally substituted once or multiple times by a group or atom selected from chlorine, fluorine, bromine, iodine, nitro, and optionally C. 1-4 Alkyl substitution once or twice for amino, cyano, azide, C 1-4 Alkyloxy, trifluoromethoxy, and trifluoromethyl;

[0067] R8 is hydrogen, C 1-6 Alkyl, OR11 or NHCOR 10 , where R 11 It is hydrogen, C 1-6 Alkyl, formyl, C 1-6 Alkyl, aryl or aryl-C 1-6 Alkyl and R 10 It is hydrogen, C 1-6 Alkyl, C 1-6 Alkoxy, mono- or di-C 1-6 Alkylamino, amino-C 1-6 Alkyl, hydroxy-C 1-6 Alkyl, Halogenated -C 1-6 Alkyl, C 1-6 Acyloxy-C 1-6 Alkyl, C 1-6 Alkoxycarbonyl-C 1-6 -alkyl, aryl, or heteroaryl; the R8-N-CO-R7 group is cis relative to the R5 group; and X is oxygen or NR. 12 , where R 12 Is it hydrogen or C? 1-6 alkyl;

[0068] Formula II

[0069]

[0070] in

[0071] Q is O or the formula = NHOR 43 Oxime, of which R 43 yes

[0072] (i) Selected from H, C 1-4 Fluorinated alkyl groups or optionally substituted C 1-4 Alkyl, or

[0073] (ii)-A 300 -R 300 A 300 It is a direct bond, -C(O)O*, -C(R3)(R4)O*, -C(O)OC(R3)(R4)O*-, or -C(R3)(R4)OC(O)O*, where the atom marked with * is directly connected to R. 300 R3 and R4 are independently selected from H, fluorine, and C. 1-4 Alkyl or C 1-4 Fluoroalkyl groups, or R3 and R4 together with the atoms they are attached to, form cyclopropyl groups, and R... 300 Selected from groups [1], [2], [2A], [3], [4], [5], or [6];

[0074] R2 is H.

[0075] A is a direct bond, -C(O)O*, -C(R3)(R4)O*, -C(O)OC(R3)(R4)O*-, or -C(R3)(R4)OC(O)O*, where the atom marked with * is directly connected to R1, and R3 and R4 are independently selected from H, fluorine, and C. 1-4 Alkyl or C 1-4 Fluoroalkyl groups, or R3 and R4 together with the atoms they are attached to, form cyclopropyl groups.

[0076] R1 is selected from groups [1], [2], [2A], [3], [4], [5], and [6], wherein the atom marked with ** is directly connected to A:

[0077]

[0078] R5 and R6 are each independently selected from H and C. 1-4 Alkyl, C 1-4 Fluorinated alkyl groups and

[0079] benzyl;

[0080] R7 is independently selected from H and C. 1-4 Alkyl and C 1-4 Fluorinated alkyl groups;

[0081] R8 is selected from:

[0082] (i)H, C 1-4 Alkyl or C 1-4 Fluorinated alkyl groups, or

[0083] (ii) Natural or non-natural α-amino acids or peptide mimics or other peptide side chains as described herein, or

[0084] (iii) Biotin or chemically linked to biotin;

[0085] R9 is selected from H, -N(R) 11 (R) 12 ) or -N + (R 11 (R) 12 (R) 13 )X - or -N(R) 11 )C(O)R 14

[0086] Where R 11 R 12 and R 13 Independently selected from H and C 1-4 Alkyl or C 1-4

[0087] Fluoroalkyl,

[0088] R 14 It is H, C 1-4 Alkyl or C 1-4 Fluoroalkyl,

[0089] R 15 Selected independently from C 1-4 Alkyl and C 1-4 Fluoroalkyl groups, and

[0090] X - It is a pharmaceutically acceptable anion.

[0091] The term "comprising," synonymous with "including," "containing," or "characterized in," is inclusive or open-ended and does not exclude additional unlisted elements or components (or steps, in the case of a method) from the drug. The phrase "consisting of" excludes any elements, steps, or components (or steps, in the case of a method) not specified in the drug. The phrase "consisting substantially of" indicates the specified materials and those (or steps, in the case of a method) that do not substantially affect the essential and novel characteristics of the drug. The essential and novel characteristics of the invention are described in this specification and include the following capabilities of the drugs and methods of the invention: blocking or modulating the junctional protein gap connecting hemichannels and preserving, protecting, restoring, or rescuing retinal structures, preserving, protecting, restoring, or rescuing choroidal structures, preserving, protecting, restoring, or rescuing retinal function, and preserving, protecting, restoring, or rescuing choroidal function, as appropriate. Substantial changes to the fundamental and novel features of this invention (including the pharmaceuticals and methods described herein) include undesirable or clinically unwelcome, harmful, adverse, or detrimental reductions in hemichannel modulation and / or the preservation, protection, restoration, or rescue of retinal structures, choroidal structures, retinal functions, and choroidal functions. In one embodiment, the pharmaceuticals comprise a connexin 43 hemichannel blocker, such as a small molecule connexin 43 hemichannel blocker, substantially composed of or consisting of therein.

[0092] In another aspect, the present invention provides the use of hemichannel blockers in the preparation of medicaments (or packages or kits containing one or more medicaments and / or containers, with or without instructions for use), said medicaments for modulating hemichannels and treating any diseases, disorders, and / or conditions described or mentioned herein. In one aspect, for example, the present invention provides the use of small molecule connexin hemichannel blockers (including, for example, Xiflam and / or its analogues or prodrugs). In one embodiment, said medicament comprises a connexin 43 hemichannel blocker, for example, a small molecule connexin 43 hemichannel blocker, substantially composed of or consisting of therein. In one embodiment, hemichannel blocker compositions useful in the present invention may comprise pharmaceutically acceptable carriers and may be formulated as, for example, pellets, solutions, microspheres, liposomes, nanoparticles, implants (including, for example, peritoneal, subcutaneous, and ocular implants, as well as sustained-release or controlled-release implants), matrix, or hydrogel formulations, or may be provided in a low-pressure lyophilized form.

[0093] The hemichannel regulated for the purposes described herein can be any connexin of interest for this purpose. For example, the hemichannel regulated for the purposes described herein can be a connexin hemichannel expressed in the retina, blood vessels, and / or vessel walls. In one embodiment, the hemichannel blocker blocks connexin hemichannels in blood vessels. In other embodiments, the hemichannel blocker blocks connexin hemichannels in blood microvessels. In other embodiments, the hemichannel blocker blocks connexin hemichannels in capillaries. In other embodiments, the hemichannel blocker blocks connexin hemichannels in the endothelium.

[0094] In different implementations, as an example, the regulated hemichannel includes one or more of connexin 36 (Cx36), connexin 37 (Cx37), connexin 40 (Cx40), connexin 43 (Cx43), connexin 45 (Cx45), connexin 57 (Cx57), connexin 59 (Cx59) and / or connexin 62 (Cx62).

[0095] In one embodiment, particularly when it involves the retina, the regulated hemichannel comprises one or more of the proteins Cx36, Cx37, Cx40, Cx43, Cx45, or Cx57. Targeted hemichannel connective proteins include, for example, one or more of the following: selected hemichannel connective proteins in blood vessels (e.g., Cx37, Cx40, or Cx43), and hemichannel connective proteins in astrocytes (e.g., Cx43), amacrine cells (e.g., Cx36, Cx45), bipolar cells (e.g., Cx36, Cx45), outer and inner plexiform layers, ganglion cell layers (e.g., Cx36, Cx45), cone photoreceptors, retinal endothelial cells, and other retinal neurons. In some embodiments, Cx36 and Cx43 hemichannels are targeted. In a particular embodiment, the hemichannel and / or the regulated hemichannel comprises Cx43. In one embodiment, the cells targeting the outer plexus layer contain hemichannels of connecting proteins (e.g., Cx43), wherein the method of the present invention can stop and reverse OPL thinning and rescue OPL.

[0096] In other embodiments, particularly those relating to choroidal or retinal vessels, the regulated hemichannel may preferably contain one or more of Cx37, Cx40, or Cx43 proteins. In one particular embodiment, the hemichannel and / or the regulated hemichannel contains Cx43. In one embodiment, a hemichannel containing vascular connexins is targeted in the outer choroidal cells, also known as Haller's layer, which consists of large-bore, non-porous vessels. In another embodiment, a hemichannel containing vascular and endothelial cell connexins is targeted in the inner choroidal cells, also known as Sattler's layer, which consists of significantly smaller vessels. In another embodiment, hemichannels containing connexins are targeted in both the outer and inner choroidal cells. In another embodiment, hemichannels containing connexins are targeted in the capillaries of the choroidal capillary layer. In one embodiment, the hemichannel vascular connexins targeted in the method of the present invention include hemichannel connexins in pericytes and connexins in vascular smooth muscle and endothelial cells. In another embodiment, the hemichannel vascular connexins targeted in the method of the present invention include hemichannels in pericytes and connexins in endothelial cells (e.g., in microcapillaries). The Cx43 hemichannel is a preferred target of the present invention.

[0097] Another embodiment of this aspect of the invention provides a drug package comprising a small molecule or other half-channel blocker. In one embodiment, the half-channel blocker is Xiflam (tornaboza).

[0098] In another embodiment, the hemichannel blocker comprises or is substantially composed of peptide 5, GAP9, GAP19, GAP26, GAP27 or α-connector protein carboxyl terminus (ACT) peptide (e.g., ACT-1 or other active anti-hemichannel peptide mimics).

[0099] Certain bioassays can be used to evaluate the activity of hemichannel blockers. Known or candidate hemichannel blockers can be identified, evaluated, or screened for their effects on molecular motility using the methods described in the examples below or other known or equivalent methods in the art for determining the effects of compounds on protein hemichannels. Various methods known in the art, including dye transfer assays, such as the transfer of molecules labeled with detectable markers, and transmembrane channels with small fluorescent permeable tracers, have been widely used to study the functional state of hemichannels. Various embodiments of this aspect of the invention are described herein, including methods for identifying or evaluating the ability of compounds to block hemichannels, comprising: (a) placing a test sample and a test system together, the test sample comprising one or more test compounds, and the test system comprising a system for evaluating hemichannel blockade, characterized in that it exhibits, for example, increased transfer of dyes or labeled metabolites, in response to the introduction of hypoxia or ischemia into the system, inflammatory mediators, or other compounds or events that induce hemichannel opening, such as extracellular Ca2+. 2+ (a) a decrease in the amount of a substance; and (b) determining the presence or increase of a metabolite, such as a dye or other labeled substance, in the system. Positive and / or negative controls may also be used. Optionally, a predetermined amount of a half-channel blocker (e.g., Xiflam) may be added to the assay system. Other methods that can be used to evaluate the activity of half-channel blockers include electrophysiological and channel conduction blocking techniques, reduction of cytoplasmic swelling or cell edema, and reduction of cellular potassium efflux, all of which are known in the art.

[0100] In one aspect, methods are provided for using assays (including assays using ARPE-19 cells) to identify, measure, or evaluate the activity of compounds that can be used to restore or salvage retinal function. See Dunn KC, et al., ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. ExpEye Res. Feb. 1996; 62(2): 155-69. Existing methods can be used to identify, measure, or evaluate the activity of compounds that can be used to restore or salvage choroidal structure and function. For example, choroidal thickness can be measured using ultrasound, magnetic resonance imaging (MRI), and enhanced depth imaging optical coherence tomography (EDI-OCT). EDI-OCT is a non-invasive method that can image the retina and choroid in cross sections and has been used to measure choroidal thickness with acceptable reproducibility and sensitivity. Choroidal thickness has been shown to be positively correlated with retinal function, with thicker choroid associated with better retinal function, as measured, for example, by multifocal electroretinography (mfERG). Other retinal-choroidal anatomical evaluation methods can be used to identify, measure, or evaluate the activity of compounds that can be used to restore or salvage choroidal function, including scanning source OCT (SS-OCT). Attached Figure Description

[0101] Figure 1 shows the raw ECG waveforms (A) of animals treated with the mediator or drug; the effects of tonabosa, the mediator, and the hemichannel modulator (tonabosa) at 0.26 mg / kg (B and E), 0.8 mg / kg (C and F), and 2.4 mg / kg (D and G) on the mixed a- and b-wave amplitudes of the ERG. The mediator data shown are from 2 weeks post-injury; in these animals, ERG function did not recover. Statistical analysis was performed using two-way ANOVA and Bonferroni post-hoc test. Significance values ​​are indicated by an asterisk: *p < 0.05; **p < 0.01; ***p < 0.001.

[0102] Figure 2 shows the effects of the medium and 2.4 mg / kg of the semi-channel modulator (tonabosa) on the mixed a-wave (A) and b-wave (B) amplitudes of the ERG in light-damaged rats after 3 months of treatment. Analysis of rod PIII (C) and PII (D) showed that untreated animals had significantly reduced amplitudes compared to pre-light-damage levels. Treated animals maintained retinal function, matching the control group for rod PII, while rod PIII was only slightly lower. All mean data are presented as mean ± SEM. Statistical analysis of a- and b-wave values ​​was performed using two-way ANOVA and Bonferroni post-hoc tests. Statistical analysis of rod PII and PIII was performed using unpaired t-tests with Welch correction. Significance is indicated by an asterisk: ***p < 0.001. LD = light damage.

[0103] Figure 3 shows the effect of oral delivery of the semichannel modulator (tonaboxa) on retinal and choroidal thickness in light-damaged rats. Baseline images and optical coherence tomography (OCT) images are shown for normal Sprague Dawley (SD) rats (A), animals treated with 2.4 mg / kg tonaboxa at 2 weeks post-light damage (B), and light-damaged rats treated with the mediator (C). Green lines on the baseline images indicate the scan location of adjacent cross-sectional OCT images. Colored lines on the OCT images highlight the internal limiting membrane (anthocyanins), OPL (orange), ONL (orange to yellow), and choroid (green to red). Quantitative analysis showed that, compared to normal (before light exposure), the mediator-treated animals exhibited thinning of both the ONL and choroid at 2 weeks post-light damage. At any time point analyzed, at 24 hours, 1 week, and 2 weeks post-light damage (DF row), no thinning of the ONL or choroid was observed in animals treated with any of the three tonaboxa doses used. Although some thinning was observed at the lowest and intermediate doses, it was not significant. ** = p < 0.01; *** = p < 0.001. Proportion bars = 100 μm

[0104] Figure 4 shows the effects of mediator (A) or treatment with a semi-channel modulator (tonaboxazole, 2.4 mg / kg) 3 months after light damage (B). Representative OCT images show significant thinning in the mediator-treated animals, particularly in the INL, ONL, and choroid. Color lines on the OCT images highlight the internal limiting membrane (anthocyanins), INL (orange to yellow), ONL (yellow to red), choroid (red to purple), and sclera (purple to green). Measurements of INL, ONL, and choroid thickness are shown in CE for the retina before damage, 3 months after LD treatment with mediator, and 3 months after LD treatment with tonaboxazole. Data are presented as mean ± SEM. Significance compared to the mediator (peanut butter) group with light damage is indicated by an asterisk: *p < 0.05; **p < 0.01; ***p < 0.001. LD = light damage. Scale bars = 100 μm

[0105] Figure 5 shows the immunohistochemical analysis of the effects of three concentrations of the orally delivered hemichannel modulator (tonabosa) on photodamaged rats. Compared to the mediator group (A), orally treated rats showed less connein 43 immunoreactivity in the retina at all three dose levels (BD). Compared to mediator-treated rats (E), Iba-1 immunolabeled cells showed low activation (budding) in the IPL of the retina of tonabosa-treated rats at all three doses (FH), although a slight increase in Iba-1 reactivity was evident at the lowest oral dose of 0.26 mg / kg. Compared to mediator-treated rats (I), GFAP immunoreactivity was not increased in the retina at 0.8 mg / ml (K) and 2.4 mg / kg (L). At the lowest oral dose of 0.26 mg / kg, GFAP labeling was slightly increased, but its expression remained lower than that of the mediator alone (J). Abbreviations: CGL: Ganglion cell layer; plexiform layer within the IPL. Scale bar: 50 μm.

[0106] Figure 6 shows the quantification of GFAP immunoreactivity area (A), connexin 43 expression (B), and mean number of Iba-1 activated cells (C) in each of the three oral tonabosar dose levels in animals treated with the mediator alone, compared to photodamaged rats. Analysis revealed significantly reduced upregulation of GFAP and connexin 43 in all three tonabosar treatment groups compared to the mediator alone (p < 0.001) (AB). Quantification of Iba-1 positive cells revealed a significantly reduced number of active microglia in all three tonabosar treatment groups compared to the mediator alone (p < 0.001) (C). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test. Significance compared to the untreated group is indicated by an asterisk: ***p < 0.001

[0107] Figure 7 shows representative OCT images of hyperglycemic rats, showing an average of 5–8 hyperreflectance points per eye (based on 7 evenly distributed OCT scans on the retina, thus underestimating the whole eye), which were absent in normal SD rats (A). The hyperreflectance points appear to be microaneurysms (less than 20 μm in diameter; arrow in B0) and large aneurysms (140–160 μm; arrow in C), specifically located in the INS and ONL. Color lines on the OCT images highlight the INL (orange to yellow), ONL (yellow to red), and choroid (purple to cyan). Evans blue dye perfusion confirmed vascular leakage at the aneurysm site mapped using OCT. The green line on the base image (D) shows the location of the OCT scan (E). The hyperreflectance points (arrows) are microaneurysms. Injection of Evans blue into the rats, followed by retinal removal and imaging of the area, reveals the vascular leakage area (F). Leakage was not present in all microaneurysms, but was consistently observed in four hyperglycemic rats with microaneurysms. Scale bar = 100μm

[0108] Figure 8 shows the ERG analysis of retinal function in hyperglycemic rats at 5 weeks postnatal compared to normal SD rats from a derived hyperglycemic strain. Representative mixed a- and b-waveforms of the ERG are shown in A and B. The mean mixed a-wave amplitude was significantly reduced in hyperglycemic rats compared to normal SD rats. The mixed b-wave amplitude was also significantly reduced in hyperglycemic rats compared to normal SD rats. Decomposition analysis showed that the amplitude was significantly reduced in hyperglycemic rats for rod PIII (C), PII (D), cone PII (E) responses and for the OP control group. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test. Significance compared to normal SD rats is indicated by an asterisk: **p < 0.01; ***p < 0.001. OP = oscillatory potential.

[0109] Figure 9 shows OCT and ERG analyses of retinal structure and function in hyperglycemic rats at 8 weeks compared to vector-treated animals (at the lowest dose used, 0.28 mg / kg), once daily for 14 days (weeks 5–7). (A) High reflectivity points are barely visible after treatment (B). The ERG of treated hyperglycemic rats was significantly restored compared to vector-treated rats, while it further deteriorated in untreated rats from week 5 to week 8. At week 8, mixed a-waves were significantly higher in treated animals compared to vector-treated animals (C). Similarly, mixed b-waves were significantly restored in animals treated with tonaboxavar at all intensities compared to the vector control group (D). Further analysis showed significant restoration of rod PIII (E), PII (F), cone PII (G), and cumulative OP (H) amplitudes in treated hyperglycemic rats. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test. Significance values ​​compared to mediator therapy are indicated by an asterisk: **p < 0.01; ***p < 0.001. OP = Oscillation potential. Scale bars = 100 μm

[0110] Figure 10 shows immunohistochemical markers in tonaboxa-treated and vector-treated hyperglycemic rats at 8 weeks of age. Strong GFAP marking was observed in the CGL, where astrocytes were located in the area surrounding microaneurysms in the retina of hyperglycemic rats, extending from the neurofibrillary layer to the ONL, indicating Müller cell activation (A). Abnormally high Iba-1 marking was present in the hyperglycemic retina of the IPL (B), with cells exhibiting enlarged cell bodies and numerous elongated branches, and connexin 43 marking was abnormally high in the GCL of untreated animals (C). Hyperglycemic rats fed tonaboxa daily for 14 days exhibited reduced inflammation, as evidenced by the marking of all three markers (DF). Quantification of the results in GI showed that all three markers—GFAP, connexin 43, and Iba-1—were significantly elevated in vector-treated rats compared to the undamaged control retina, while tonaboxa treatment resulted in a significant reduction in marking at 8 weeks, and significantly lower levels in the retina of untreated rats. Statistical analysis was performed using one-way ANOVA, followed by Tukey's multiple comparison test. Significance compared to the untreated group is indicated by an asterisk: ***p < 0.001. Proportion bars = 100 μm. Detailed Implementation

[0111] Increased connexin 43 hemichannel opening is associated with activation of inflammasome pathways and inflammation in a range of pathologies, including ocular disorders. Using animal models of light-damaged retina in dry AMD and a spontaneous rat model of DR, we have identified the efficacy of clinically safe doses of connexin hemichannel blockers (such as orally delivered small molecule connexin hemichannel blockers, including Xiflam) in restoring and salvaging retinal function and morphology, as well as choroidal function and structure. Clinical parameters (basal imaging, optical coherence tomography (OCT), and electroretinography) and inflammatory markers (Iba-1 microglia marker, astrocyte marker glial fibrillary acidic protein, and connexin 43 protein expression, immunohistochemistry) were evaluated, and hemichannel blocker treatment resulted in preservation of retinal photoreceptor function when assessed up to 3 months after light damage in the dry AMD model. In the DR model, clinical signs, including the presence of aneurysms confirmed by Evans blue perfusion, decreased after two weeks of daily tonabosar treatment. Inflammation also decreased, and retinal function recovered. We have discovered that semichannel blockers can be used to not only improve but also restore anatomical and functional outcomes in chronic retinal diseases.

[0112] Surprisingly, a single oral dose of a hemichannel blocker was found to have neuroprotective effects during a 3-month evaluation period following assessment of acute photodamage. Hemichannel blocker treatment was found to significantly preserve retinal function, particularly the function of photoreceptors and bipolar cells in the inner retina. Furthermore, the determined increase in oscillatory potential using each of the three hemichannel blocker doses studied indicates a protective effect on inner retinal cells despite photodamage. Improved PIII and PII responses in electroretinography (ERG) also demonstrate the specific preservation of phototransduction pathways and post-photoreceptor neuronal responses. This study, described in Example 2, also demonstrates the use of hemichannel blockers to preserve retinal layer structures, as measured by OCT. Further details regarding these findings can be found in Example 2.

[0113] Furthermore, as shown in Example 3, it was found that semi-channel blockers (e.g., oral blockers, such as Xiflam) effectively blocked signs of diabetic DR secondary to spontaneous and chronic systemic hyperglycemia in a diabetic SD rat model. Signs of microaneurysms and large aneurysms in the retina were observed in this phenotypic model of diabetes and DR, accompanied by effects on visual retinal function.

[0114] Compared to placebo control, the application of an oral hemichannel blocker (in this case, Xiflam) demonstrated regression of microaneurysms and large aneurysms and significant salvage of retinal function, as measured by ERG. The results from these entirely different models have implications for other chronic ocular inflammatory diseases, particularly those involving inflammasome pathways. The hemichannel blockade in the macular degeneration and diabetic retinopathy models described in this patent not only reduced inflammation but also surprisingly preserved and salvaged retinal structure and function, as well as the vital choroidal structures.

[0115] This application relates to a remarkable discovery of regulation of semi-channel opening, which has a direct and lasting effect on the maintenance and salvage of retinal structure and function, as well as choroidal structure. See Examples 1-3 below. These findings have significant implications for the treatment of various diseases, disorders, and conditions characterized by complete or partial loss of retinal structure and / or function, including diabetic retinopathy, for which there is currently no cure.

[0116] It has also been found that hemichannel blockers (including, for example, connexin 43 hemichannel blockers) can be used to preserve the choroid. Therefore, hemichannel blockers can be used as a method to maintain choroidal function in disease states.

[0117] definition

[0118] As used herein, the term “about” means a value or parameter as understood in the art and includes embodiments for that value or parameter itself. For example, a description referring to “about X” includes a description of “X”. For example, the weight value of the term “about 5 mg” in dosage means + / - 0.5 degrees of said weight value.

[0119] “Small molecule” is defined herein as having a molecular weight of less than about 600 to 900 Daltons and is typically an organic compound. Small molecules can be active agents of half-channel blocker prodrugs. In one embodiment, the small molecule has a molecular weight of less than 600 Daltons. In another embodiment, the small molecule has a molecular weight of less than 900 Daltons.

[0120] As used herein, “treatment” (and its grammatical variations such as “treat” or “treating”) refers to a clinical intervention to alter the natural processes of an individual, tissue, or cell being treated, and may be performed for prevention or during clinicopathological procedures. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, disorder, or condition; alleviating signs or symptoms; reducing any direct or indirect pathological consequences of the disease; slowing the rate of disease progression; improving or alleviating the disease state; and reducing or improving prognosis. In some embodiments, the compounds, methods, and compositions of the present invention may be used to delay the development of a disease, disorder, or condition, or to slow the progression of a disease, disorder, or condition. This term does not necessarily mean treating a subject until complete recovery. Therefore, “treatment” includes reducing, alleviating, or improving the symptoms or severity of a particular disease, disorder, or condition, or preventing or otherwise reducing the risk of developing a particular disease, disorder, or condition. It may also include maintaining or promoting a complete or partial remission of the condition.

[0121] As used herein, "treatment" also includes the preservation and / or salvage of retinal structure, retinal function, choroidal structure, and / or choroidal function in subjects following the administration of a hemichannel blocker. A preferred hemichannel blocker is Xiflam. The preferred route of administration is oral.

[0122] The term "treatment" of a disease, symptom, or disorder can refer to the prevention, mitigation, reduction, and especially the cessation and reversal of the disorder, disease, or symptom, and / or the improvement and salvage or restoration or normalization of retinal structure and / or function, and / or the improvement and salvage or restoration or normalization of choroidal structure and / or function. Specifically, for example, in stopping or reversing a disorder, disease, or symptom, or in salvaging retinal function and / or structure, or in salvaging choroidal function and / or structure, one or more or all symptoms of the disorder, disease, or symptom are reversed or substantially eliminated; the ONL in the retina is salvaged, restored, and / or normalized; retinal ERG function, inner retinal function, retinal photoreceptor function (especially rod photoreceptor function) and / or retinal PIII and PII rod responses are salvaged, restored, and / or normalized; and the choroidal capillary layer in the choroid is salvaged, restored, and / or normalized.

[0123] In other embodiments, the compounds and methods described herein are used to protect the outer and inner nuclear layers of the retina, as shown in the examples. This is important in chronic retinal diseases, including age-related macular degeneration, where the protective effects of the present invention are also found to be effective.

[0124] The term "prevention" refers to the complete or partial prevention, improvement, or control of something.

[0125] As used herein, “effective amount” means the amount that effectively achieves the desired therapeutic or preventative outcome at the necessary dose and for the necessary duration. For example, and without limitation, “effective amount” may mean the amount of a compound or composition disclosed herein that is capable of treating signs and / or symptoms of a disease, disorder, or condition involving damaged retinal and / or choroidal structure and / or function, or the amount of a semichannel compound or composition that can beneficially modulate and salvage damaged retinal and / or choroidal structure and / or function.

[0126] As used herein, the “therapeutic effective amount” of the substance / molecule, agonist, or antagonist of the present invention can vary depending on factors such as an individual’s disease state, age, sex, and weight, and the ability of said substance / molecule, agonist, or antagonist to elicit a desired response in the individual. Therapeutic effective amount is preferably also an amount in which the beneficial therapeutic effect may outweigh any toxic or harmful effects of said substance / molecule, agonist, or antagonist. A therapeutically effective amount of a semichannel blocker will beneficially maintain or improve retinal structure and / or function, and / or maintain or improve choroidal structure and / or function in a subject.

[0127] As used in this article, "preventative effective dose" refers to the amount effective within the necessary dosage and time period to achieve the desired preventative outcome, typically maintaining salvaged or restored retinal and / or choroidal function and / or structure. Generally, but not always, the preventative effective dose will be less than the therapeutic effective dose.

[0128] The term "pharmaceutical formulation" refers to an article in a form in which the biological activity of the active ingredient contained therein (e.g., a half-channel blocker) is effective, and which does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation will be administered.

[0129] As used in this article, "pharmaceuticalally acceptable carrier" refers to any component of a pharmaceutical preparation other than the active ingredient that can be safely administered to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffer solutions, excipients, stabilizers, and preservatives.

[0130] As used herein, the term "subject," etc. (including "individual" and "patient," which are used interchangeably herein) refers to any mammal, including humans, domesticated animals, and farm animals, as well as zoo, wildlife park, sports field, or pet animals such as dogs, horses, cats, sheep, pigs, cattle, etc. Preferred mammals are humans, including adults, children, and the elderly. Preferred sports field animals are horses and dogs. Preferred pet animals are dogs and cats. In some embodiments, the subject, individual, or patient is a human.

[0131] As used in this article, the term "half-channel" refers to part of a gap junction (two hemichannels or conjoints that connect the intercellular spaces between adjacent cells to form a gap junction) and contains numerous conjoint proteins, typically homo- or hetero-hexamers of conjoint proteins, that form pores in the gap junction between the cytoplasm of two adjacent cells. The half-channel is provided by the cell on one side of the junction, and two half-channels from opposing cells typically merge to form a complete intercellular half-channel. However, in some cells, and in certain conditions, the half-channel itself is active as a conduit between the cytoplasm and the extracellular space, thus allowing the transfer of ions and small molecules.

[0132] Compounds of Formula I, such as Xiflam, and / or any analogues or prodrugs of the aforementioned compounds, can modulate the function and / or activity of hemichannels, preferably those comprising any type of connexin. Therefore, the reference to “hemichannel” should be broadly understood to include hemichannels that comprise, are substantially composed of, or are composed of, any of a variety of different connexins, unless the context otherwise requires. However, by way of example, a hemichannel may comprise one or more of any connexins, including those specifically mentioned above. In one embodiment, the hemichannel comprises one or more of the aforementioned connexins. In one embodiment, the hemichannel comprises one or more of connexins 36, 37, 40, 43, 45, and 57. In one embodiment, the hemichannel comprises one of connexins 37, 40, or 43. In one embodiment, the hemichannel is a connexin 43 hemichannel. In one embodiment, the hemichannel is a retinal hemichannel. In one embodiment, the hemichannel is a choroidal hemichannel. In one embodiment, the hemichannel is a vascular hemichannel. In one embodiment, the hemichannel is a connexin hemichannel found in vascular endothelial cells. In one specific embodiment, the hemichannel comprises one or more of connexins 30, 37, and 43. In one specific embodiment, the hemichannel is composed of connexin 30. In one specific embodiment, the hemichannel is composed of connexin 37. In one specific embodiment, the hemichannel is composed of connexin 43. In one embodiment, the hemichannel comprises one or more connexins, excluding connexin 26. In one embodiment, the composition may include or exclude any connexin hemichannel blocker, including those described above.

[0133] Hemichannels and semichannels can be present in any type of cell. Therefore, references to “halfchannel” or “semichannel” should be understood to include references to halfchannels or semichannels present in any cell type, unless the context requires otherwise. In one embodiment of the invention, a halfchannel or semichannel is present in cells of an organ, or in cancer or tumor. In one embodiment, the halfchannel is a vascular halfchannel. In one embodiment, the halfchannel is a connective protein halfchannel found in vascular endothelial cells and / or vascular smooth muscle cells, or in the retina and / or choroid or choroidal vascular system.

[0134] As used herein, "modulation of half-channels" refers to the regulation of one or more functions and / or activities of half-channels, typically the flow of molecules between cells through half-channels. Such functions and activities include, for example, the inflow of molecules from the extracellular space or environment into the cell through half-channels, and / or the inflow of molecules from the intracellular space or environment into the extracellular space or environment through half-channels. Compounds that can be used to modulate half-channels may be called "half-channel modulators." All aspects of the inventions and methods described herein can be achieved through the regulation of half-channels.

[0135] Regulation of hemichannel function can occur in any manner. However, by way of example only, regulation can occur through one or more of the following: inducing or promoting hemichannel closure; preventing, blocking, inhibiting, or reducing hemichannel opening; triggering, inducing, or promoting the internalization of hemichannels and / or gap junctions. The use of terms such as “block,” “inhibit,” “prevent,” “reduce,” and “antagonize” may not imply complete blockage, inhibition, prevention, or antagonism, although this may be preferred, and should be understood to include partial blockage, inhibition, prevention, or antagonism to at least reduce the function or activity of hemichannels and / or gap junctions. Similarly, “inducing” or “promoting” should not be construed as implying complete internalization of hemichannels (or groups of hemichannels), but rather as including partial internalization to at least reduce the function or activity of hemichannels.

[0136] As used herein, the terms “anti-half-channel compounds” and “half-channel blockers” are compounds that interfere with the passage of molecules through junctional protein half-channels. Anti-half-channel compounds or half-channel blockers can block or reduce half-channel opening, block or reduce the release of molecules through the half-channel into the extracellular space, and / or block or reduce the entry of molecules through the half-channel into the intracellular space. Anti-half-channel compounds and half-channel blockers include compounds that completely or partially block half-channel leakage or the movement of molecules into or out of the extracellular space. Anti-half-channel compounds and half-channel blockers also include compounds that reduce the probability of half-channel opening. The probability of opening is a measure of the percentage of time a channel remains open relative to the time it is closed (see Goldberg GS, et al., Selective permeability of gap junction channels Biochimica et Biophysica Acta 1662 (2004) 96-101 for a review). Anti-half-channel compounds and half-channel blockers include half-channel regulators. Anti-half-channel compounds and half-channel blockers may directly or indirectly interfere with the passage of molecules through junctional protein half-channels. All aspects of the inventions and methods described herein can be achieved, for example, by blocking or reducing the probability of opening a hemichannel as described herein. In one embodiment, the connexin hemichannel is a connexin 43 hemichannel and / or other vascular connexin hemichannels.

[0137] As used in this article, the terms "restoring or salvaging retinal structure," "saving or restoring retinal structure," and "saving and / or restoring retinal structure," etc., refer to improving the integrity of retinal structures, including, for example, the restoration of retinal pigment epithelium, the restoration of retinal vascular endothelium, and / or the restoration of normal retinal layer structures. The terms "restoring or salvaging retinal structure," etc., also refer to reducing or eliminating microaneurysms and / or large aneurysms (see, for example, Figure 9B In some embodiments of the invention, retinal structures are salvaged and restored to a normal or pre-disease state. In some embodiments of the invention, the retinal pigment epithelium, retinal vascular endothelium, and / or retinal layer structures are salvaged and restored to a normal or pre-disease state.

[0138] The terms "restoring or salvaging retinal function" and "saving or restoring retinal function," "saving and / or restoring retinal function," etc., refer to improving retinal function, including, for example, improving the mixed a-wave function (see, for example, Figure 9C ), improve the mixed b-wave function (see, for example, Figure 9D ) and / or improve PII and PIII rod and cone function (see, for example, Figure 9E-G), which can be evaluated, for example, by electroretinography. The terms "restoration or salvage of retinal function," etc., also refer to improvement of overall ERG function. See also Figure 1, which shows the salvage of ERG function and inner retinal function, and Figure 2, which shows the improvement of photoreceptor function. In some embodiments of the invention, retinal function is salvaged and restored to a normal or pre-disease state. In some embodiments of the invention, retinal ERG, PII and PIII rod and / or cone functions, etc., are salvaged and restored to a normal or pre-disease state.

[0139] As used herein, the terms "restoring or salvaging the choroidal structure," "saving or restoring the choroidal structure," and "saving and / or restoring the choroidal structure," etc., refer to improving the integrity of the choroidal structure, including, for example, the restoration of choroidal thickness and / or the restoration of the choroidal vascular bed, which can be determined, for example, using OCT angiography or fluorescein fluorophore angiography. In some embodiments of the invention, the choroidal structure is salvaged and restored to a normal or pre-disease state. In some embodiments of the invention, choroidal thickness and / or the choroidal vascular bed is salvaged and restored to a normal or pre-disease state.

[0140] As used herein, the terms "restoring or salvaging choroidal function," "saving or restoring choroidal function," "saving and / or restoring choroidal function," etc., refer to improvements in choroidal blood flow, which can be determined, for example, using high-speed OCT angiography. The terms "restoring or salvaging choroidal function," etc., also refer to improvements in choroidal vessel blood flow to the outer retina and improvements in the regulation of choroidal blood flow. In some embodiments of the invention, choroidal function is salvaged and restored to a normal or pre-disease state. In some embodiments of the invention, choroidal blood flow is salvaged and restored to a normal or pre-disease state.

[0141] The compounds of this invention can be used in treatments to preserve or salvage retinal structure, retinal function, choroidal structure and / or choroidal function, including in treatments for diseases, disorders, or conditions characterized wholly or partially by a pathological, abnormal, or otherwise undesirable or unwelcome reduction in the integrity of retinal and / or choroidal structure or function. The integrity of the retina and / or choroid is crucial for preventing vision loss.

[0142] The terms "peptide," "peptide mimic," and "analyst" include synthetic or genetically engineered chemical compounds that may have substantially the same structural and functional characteristics as the protein regions they mimic. In the case of hemichannel connective proteins, these can be mimicked, for example, the extracellular loop of hemichannel connective proteins.

[0143] This patent describes novel methods for preserving or salvaging retinal structures, retinal functions, choroidal structures and / or choroidal functions, which can be improved in many diseases, disorders or conditions, some of which are characterized by chronic retinal dysfunction and / or loss of retinal structure and / or chronic choroidal dysfunction and / or loss of choroidal structure.

[0144] The present invention particularly provides a method for preserving or salvaging retinal structure, retinal function, choroidal structure and / or choroidal function by administering a semi-channel blocker (such as a compound of formula I, for example Xiflam, or a compound of formula II, and / or an analogue or prodrug of any of the aforementioned compounds), for the treatment of diseases, disorders or conditions characterized entirely or partially by loss of retinal structure, retinal function, choroidal structure and / or choroidal function.

[0145] In some embodiments, the present invention characterizes the use of compounds of Formula I, such as Xiflam, or compounds of Formula II and / or analogues or prodrugs of any of the foregoing compounds, to directly and immediately block the Cx43 hemichannel and result in the preservation or salvage of retinal structure, retinal function, choroidal structure and / or choroidal function. Some exemplary dosages are in the range of about 0.1 to about 5.0 mg / kg, including, for example, 0.2 to 3.0 mg / kg, or 0.2 to 2 mg / kg and 0.2 to 1.0 mg / kg, or 0.2 to 0.5 mg / kg. Some exemplary daily or other cyclic dosage ranges are about 10-250 mg per dose, including, for example, about 20-25 mg per dose, about 25-50 mg per dose, about 50-75 mg per dose, about 75-100 mg per dose, and about 100-250 mg per dose, including doses of 20, 50, 100, and 150 mg per dose.

[0146] Connecting proteins

[0147] In different embodiments, the regulated half-channel is any connexin half-channel and may or may not include the connexin 26 (Cx26) half-channel. In some embodiments, the regulated half-channel is the connexin 36 (Cx36) half-channel, the connexin 37 (Cx37) half-channel, the connexin 40 (Cx40) half-channel, the connexin 43 (Cx43) half-channel, the connexin 45 (Cx45) half-channel, and / or the connexin 57 (Cx57) half-channel. In one embodiment, the regulated half-channel comprises one or more of the Cx36, Cx37, Cx40, Cx43, Cx45, and / or Cx57 proteins. In a particular embodiment, the half-channel and / or the regulated half-channel is the Cx37 and / or Cx40 and / or Cx43 half-channel. In a particular embodiment, the half-channel and / or the regulated half-channel is the Cx30 and / or Cx43 and / or Cx45 half-channel. In one particular implementation, the half-channel and / or the adjusted half-channel is a Cx36, Cx37, Cx43, and / or Cx45 half-channel.

[0148] In some embodiments, the regulated hemichannel may or may not include any of the aforementioned connexins. In some aspects, the hemichannel blocker is a blocker of the Cx43 hemichannel, Cx40 hemichannel, and / or Cx45 hemichannel. In some preferred embodiments, the hemichannel blocker is a connexin 43 hemichannel blocker. Pharmaceutical compositions of the present invention used for any purpose characterized herein may also contain a hemichannel blocker that can inhibit or block any of the aforementioned connexin hemichannels (including homologous and heterologous hemichannels). In some embodiments, the regulated hemichannel may or may not include any of the aforementioned connexin hemichannels, or may be a heteromeric hemichannel.

[0149] In one embodiment, the hemichannel blocker used in any of the application, co-application, composition, kit, or treatment method of the present invention is a Cx43 hemichannel blocker. Other embodiments include Cx45 hemichannel blockers, Cx30 hemichannel blockers, Cx37 hemichannel blockers, Cx40 hemichannel blockers, and blockers of one or more of connector protein hemichannels or other hemichannels, which comprise or are substantially composed of or constitute of any of the other connector proteins described above or herein. Some embodiments may include or exclude any of the foregoing connector proteins or hemichannels, or others mentioned in this patent. In various embodiments, as examples, the regulated hemichannel comprises one or more of connector proteins 36, 37, 40, 43, 45, 57, 59, and / or 62.

[0150] In one embodiment, particularly when it involves the retina, the regulated hemichannel comprises one or more of the proteins Cx36, Cx37, Cx40, Cx43, Cx45, or Cx57. Targeted hemichannel connective proteins include, for example, one or more of the following: selected hemichannel connective proteins in blood vessels (e.g., Cx37, Cx40, or Cx43), and hemichannel connective proteins in astrocytes (e.g., Cx43), amacrine cells (e.g., Cx36, Cx45), bipolar cells (e.g., Cx36, Cx45), outer and inner plexiform layers, ganglion cell layers (e.g., Cx36, Cx45), cone photoreceptors, retinal endothelial cells, and other retinal neurons. In some embodiments, Cx36 and Cx43 hemichannels are targeted. In a particular embodiment, the hemichannel and / or the regulated hemichannel comprises Cx43. In one embodiment, the cells targeting the outer plexus layer contain hemichannels of connecting proteins (e.g., Cx43), wherein the method of the present invention can stop and reverse OPL thinning and rescue OPL.

[0151] In other embodiments, particularly those relating to choroidal or retinal vessels, the regulated hemichannel may preferably contain one or more of Cx37, Cx40, or Cx43 proteins. In one particular embodiment, the hemichannel and / or the regulated hemichannel contains Cx43. In one embodiment, a hemichannel containing vascular connexins is targeted in the outer choroidal cells, also known as Haller's layer, which consists of large-bore, non-porous vessels. In another embodiment, a hemichannel containing vascular and endothelial cell connexins is targeted in the inner choroidal cells, also known as Sattler's layer, which consists of significantly smaller vessels. In another embodiment, hemichannels containing connexins are targeted in both the outer and inner choroidal cells. In another embodiment, hemichannels containing connexins are targeted in the capillaries of the choroidal capillary layer. In one embodiment, the hemichannel vascular connexins targeted in the method of the present invention include hemichannel connexins in pericytes and connexins in vascular smooth muscle and endothelial cells. In another embodiment, the hemichannel vascular connexins targeted in the method of the present invention include hemichannels in pericytes and connexins in endothelial cells (e.g., in microcapillaries). The Cx43 hemichannel is a preferred target of the present invention.

[0152] Small molecule half-channel blockers

[0153] Examples of half-channel blockers include small molecule half-channel blockers, such as Xiflam (tonaboxa). The structure of tonaboxa (also shown in PubChem, DrugBank, and MedChemExpress) is:

[0154]

[0155] Other chemical names for Tonaboza can be found in PubChem (N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydroxonen-4-yl]-3-chloro-4-fluorobenzamide), DrugBank (N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide), and Chemical textbooks. Book)(N-((3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-1-benzopyran-4-yl)-3-chloro-4-fluorobenzamide; or 2H-benzo(B)pyran-3-ol, 6-acetyl-4-(3-chloro-4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-; or N-[(3S,4S)-6-acetyl-3,4-dihydro-3-hydroxy-2,2-dimethyl-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide).

[0156] In some embodiments, the hemichannel blocker is a small molecule other than Xiflam, such as the hemichannel blocker described in Formula I or Formula II in U.S. Patent Application Publication No. 20160177298 (filed attribution to Colin Green et al., the disclosure of which is hereby incorporated in its entirety by reference), as described above. Various preferred embodiments include the use of small molecules that block, improve, or otherwise antagonize or inhibit hemichannel opening for the treatment of the diseases, disorders, and conditions described or mentioned herein. In different embodiments, the small molecule that blocks, improves, or inhibits hemichannel opening is a prodrug of Xiflam or an analogue thereof.

[0157] In some embodiments, the present invention characterizes the use of small molecule half-channel blockers (including, for example, compounds of Formula I, such as Xiflam, and / or analogs or prodrugs of any of the foregoing compounds) for blocking Cx43 half-channels (e.g. for salvaging or restoring retinal structure, salvaging or restoring retinal function, and salvaging or restoring choroidal structure and / or function).

[0158] As an example, the semi-channel blocker Xiflam (Tonaboza) is known by the IUPAC names N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydroxonen-4-yl]-3-chloro-4-fluorobenzamide or (3S-cis)-N-(6-acetyl-3,4-dihydro-3-hydroxy-2,2-(dimethyl-d6)-2H-1-benzopyran-4-yl)-3-chloro-4-fluorobenzamide.

[0159] Another useful compound is bordinine, a porphyrin alkaloid found in Bordeaux trees and Lindera aggregata.

[0160] In one embodiment, Xiflam and / or its analogues or prodrugs are selected from compounds having formula I:

[0161]

[0162] in,

[0163] Y is C-R1;

[0164] R1 is an acetyl group;

[0165] R2 is hydrogen, C 3-8 Cycloalkyl groups, C-type carbons optionally interrupted by oxygen or substituted with hydroxyl groups 1-6 Alkyl, C 1-6 alkoxy or substituted amino carbonyl, C 1-6 alkyl carbonyl, C 1-6 alkoxycarbonyl, C 1-6 Alkyl carbonyloxy, C 1-6 Alkyl, nitro, cyano, halogen, trifluoromethyl, or CF3S; or the group CF3-A-, wherein A is -CF2-, -CO-, -CH2-, CH(OH), SO2, SO, CH2-O, or CONH; or the group CF2H-A′-, wherein A′ is oxygen, sulfur, SO, SO2, CF2, or CFH; trifluoromethoxy, C 1-6 Alkyl sulfinyl, perfluorinated C 2-6 alkylsulfonyl, C 1-6 alkylsulfonyl, C 1-6 Alkoxysulfinyl, C 1-6 Alkoxysulfonyl, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, phosphonyl, arylcarbonyloxy, heteroarylcarbonyloxy, arylsulfinyl, heteroarylsulfinyl, arylsulfonyl or heteroarylsulfonyl, wherein any aromatic moiety is optionally substituted, C 1-6 Alkyl carbonyl amino, C 1-6 alkoxycarbonylamino, C 1-6 Alkyl-thiocarbonyl, C 1-6 alkoxy-thiocarbonyl, C 1-6 alkyl-thiocarbonyloxy, 1-mercapto-C 2-7 alkyl, formyl, or aminosulfinyl, aminosulfonyl, or aminocarbonyl, wherein any amino moiety is optionally surrounded by one or two C2 groups. 1-6 Alkyl substitution, or C 1-6 Alkylsulfinylamino, C 1-6 Alkylsulfonylamino, C 1-6alkoxysulfinylamino or C 1-6 alkoxysulfonylamino, or terminally C 1-6 Alkyl carbonyl, nitro, or cyano-substituted vinyl groups, or -C(C 1-6 alkyl)NOH or -C(C 1.6 Alkyl)NNH2; or optionally with one or two C 1-6 Alkyl or C 2-7 An alkyl-substituted amino group; one of R3 and R4 is hydrogen or C. 1-4 Alkyl and the other is C 1-4 Alkyl, CF3 or CH2X a It contains fluorine, chlorine, bromine, iodine, and C. 1-4 Alkoxy, hydroxy, C 1-4 Alkyl carbonyloxy, -SC 1-4 Alkyl, nitro, optionally marked with one or two Cs 1-4 Alkyl-substituted amino, cyano or C 1-4 alkoxycarbonyl; or R3 and R4 together are optionally C 1-4 Alkyl-substituted C 2-5 Polymethylene;

[0166] R5 is C 1-6 Alkyl carbonyloxy, benzoyloxy, ONO2, benzyloxy, phenoxy or C 1-6 Alkyl groups and R6 and R9 are hydrogen, or R5 is a hydroxyl group and R6 is hydrogen or C 1-2 Alkyl group and R9 is hydrogen;

[0167] R7 is a heteroaryl or phenyl group, both of which are independently and optionally substituted once or multiple times by a group or atom selected from chlorine, fluorine, bromine, iodine, nitro, and optionally C. 1-4 Alkyl substitution once or twice for amino, cyano, azide, C 1-4 Alkyloxy, trifluoromethoxy, and trifluoromethyl;

[0168] R8 is hydrogen, C 1-6 Alkyl, OR 11 or NHCOR 10 , where R 11 It is hydrogen, C 1-6 Alkyl, formyl, C 1-6 Alkyl, aryl or aryl-C 1-6 Alkyl and R 10 It is hydrogen, C 1-6 Alkyl, C 1-6 Alkoxy, mono- or di-C 1-6 Alkylamino, amino, amino-C 1-6 Alkyl, hydroxy-C 1-6 Alkyl, Halogenated -C 1-6 Alkyl, C1-6 Acyloxy-C 1-6 Alkyl, C 1-6 Alkoxycarbonyl-C 1-6 -alkyl, aryl, or heteroaryl; the R8-N-CO-R7 group is cis relative to the R5 group; and X is oxygen or NR. 12 , where R 12 Is it hydrogen or C? 1-6 alkyl.

[0169] In some embodiments, the present invention characterizes the use of small molecule half-channel blockers (including, for example, compounds of formula II, and / or analogues or prodrugs of any of the aforementioned compounds) for blocking Cx43 half-channels (e.g., for salvaging or restoring retinal structure, salvaging or restoring retinal function, and for salvaging or restoring choroidal structure and / or function).

[0170] Formula II

[0171]

[0172] in

[0173] Q is O or the formula = NHOR 43 Oxime, of which R 43 yes

[0174] (i) Selected from H, C 1-4 Fluorinated alkyl groups or optionally substituted C 1-4 Alkyl, or

[0175] (ii)-A 300 -R 300 A 300 It is a direct-connect key, -C(O)O*, -C(R3)(R4)O*,

[0176] -C(O)OC(R3)(R4)O*- or -C(R3)(R4)OC(O)O*, where the atom marked with * is directly connected to R. 300 R3 and R4 are independently selected from H, fluorine, and C. 1-4 Alkyl or C 1-4 Fluoroalkyl groups, or R3 and R4 together with the atoms they are attached to, form cyclopropyl groups, and R... 300 Selected from groups [1], [2], [2A], [3], [4], [5], or [6];

[0177] R2 is H.

[0178] A is a direct bond, -C(O)O*, -C(R3)(R4)O*, -C(O)OC(R3)(R4)O*-, or -C(R3)(R4)OC(O)O*, where the atom marked with * is directly connected to R1, and R3 and R4 are independently selected from H, fluorine, and C. 1-4 Alkyl or C 1-4 Fluoroalkyl groups, or R3 and R4 together with the atoms they are attached to, form cyclopropyl groups.

[0179] R1 is selected from groups [1], [2], [2A], [3], [4], [5], and [6], wherein the atom marked with ** is directly connected to A:

[0180]

[0181] R5 and R6 are each independently selected from H and C. 1-4 Alkyl, C 1-4 Fluorinated alkyl groups and

[0182] benzyl;

[0183] R7 is independently selected from H and C. 1-4 Alkyl and C 1-4 Fluorinated alkyl groups;

[0184] R8 is selected from:

[0185] (i)H, C 1-4 Alkyl or C 1-4 Fluorinated alkyl groups, or

[0186] (ii) Natural or non-natural α-amino acids or peptide mimics or other peptide side chains as described herein, or

[0187] (iii) Biotin or chemically linked to biotin;

[0188] R9 is selected from H, -N(R) 11 (R) 12 ) or -N + (R 11 (R) 12 (R) 13 )X - or -N(R) 11 )C(O)R 14

[0189] Where R 11 R 12 and R 13 Independently selected from H and C 1-4 Alkyl or C 1-4

[0190] Fluoroalkyl,

[0191] R 14 It is H, C 1-4 Alkyl or C 1-4 Fluoroalkyl,

[0192] R 15 Selected independently from C 1-4 Alkyl and C 1-4 Fluoroalkyl groups, and

[0193] X - It is a pharmaceutically acceptable anion.

[0194] In some implementations, Q is O.

[0195] For any of the above-mentioned Markush groups, the group may or may not include any species listed for that group. The half-channel blocker used in the method of the present invention may or may not include any of these compounds.

[0196] In another embodiment, the analogue of Formula I is the compound carabosa (N-[(3R,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrochromene-4-yl]-4-fluorobenzamide) or trans-(+)-6-acetyl-4-(S)-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-1-benzo[3R-ol]pyran-3R-ol, hemihydrate.

[0197] In some embodiments, Xiflam and / or its analogues are in the form of a free base or a pharmaceutically acceptable salt. In other embodiments, one or more polymorphs, one or more isomers, and / or one or more solvates of Xiflam and / or its analogues may be used.

[0198] Various other small molecules have been reported for use in inhibiting half-channel activity. See Green et al., U.S. Patent Application Publication No. 20160177298, Formula II; Savory et al., U.S. Patent Application Publication No. 20160318891; and Savory et al., U.S. Patent Application Publication No. 20160318892, all of which are incorporated herein by reference in their entirety, as described above. Half-channel blockers used in the methods of the present invention may or may not include any of these compounds.

[0199] In one aspect, the present invention relates to the use, alone or in a kit, package, or other manufactured article, of a pharmaceutical composition for the treatment of the diseases, disorders, or conditions described herein, and those characterized by reduced or disordered retinal structure, retinal function, and / or choroidal structure. In some aspects, the hemichannel blocker is a connexin 43 hemichannel blocker. As mentioned above, other connexin hemichannel blockers are within the scope of this invention.

[0200] In some embodiments, a “partial precursor” refers to a substance that acts as a protecting group, masking functional groups within an active agent to convert the active agent into a prodrug. Typically, the partial precursor is linked to a drug via a bond that is cleaved in vivo in an enzymatic or non-enzymatic manner to convert the prodrug into its active form. In some embodiments, the partial precursor may also be an active agent. In some embodiments, the partial precursor may bind to a half-channel blocker molecule, peptide, antibody, or antibody fragment. In some embodiments, the partial precursor may bind to, for example, any of a peptide or peptide mimic, a small molecule, or other organic half-channel blocker. In some embodiments, the partial precursor may bind to a compound of formula I. In some embodiments, the prodrug may be another half-channel compound, for example, a compound described in U.S. Patent Application Publication No. 20160177298 by Green et al.; U.S. Patent Application Publication No. 20160318891 by Savory et al.; or U.S. Patent Application Publication No. 20160318892 by Savory et al.

[0201] Chemical delivery modification

[0202] The hemichannel blockers used in this invention can also be formulated as microparticles (microspheres, Mps) or nanoparticles (nanoparticles, Nps), or both, as well as liposomes or implants. Particulate drug delivery systems include nanoparticles (1 to 999 nm) and microparticles (1 to 1,000 μm), which are further divided into nanospheres and microspheres, and nanocapsules and microcapsules. In nanocapsules and microcapsules, drug particles or droplets are encapsulated in a polymer membrane. Particulate systems have the advantage of delivery by injection, and their size and polymer composition significantly influence their biological behavior in vivo. Compared to nanospheres, microspheres can remain within the vitreous humor for a much longer period; therefore, microparticles act as a reservoir after injection. Nanoparticles rapidly diffuse and internalize into tissues and cells.

[0203] Various methods can be used to assess the activity or potency of hemichannel blockers. In one aspect of the invention, as described herein, as an example, techniques for evaluating retinal structure, retinal function, and choroidal structure and / or function are used to evaluate or monitor the effects of hemichannel blocker treatment in subjects.

[0204] Certain bioassays can also be used to evaluate the activity of hemichannel blockers. The effects of known or candidate hemichannel blockers on molecular mobility can be identified, evaluated, or screened using the methods described in the examples below or other methods known in the art or equivalent for determining the passage of compounds through protein hemichannels. Various methods known in the art, including dye transfer assays, such as the transfer of molecules labeled with detectable markers, and transmembrane channels with small fluorescent permeable tracers, have been widely used to study the functional state of hemichannels. See, for example, Schlaper, KA, et al. Currently Used Methods for Identification and Characterization of Hemichannels. Cell Communication and Adhesion 15:207-218 (2008). In vivo methods can also be used. See, for example, the methods in the following literature: Danesh-Meyer, HV, et al. Connexin43 mimetic peptide reduces vascularleak and retinal ganglion cell death following retinal ischemia. Brain, 135:506-520 (2012); Davidson, JO, et al. (2012). Connexin hemichannel blockade improves outcomes in a model of fetal ischemia. Annals of Neurology 71:121-132 (2012).

[0205] A method for identifying or evaluating the ability of a compound to block a half-channel includes: (a) placing a test sample and a test system together, the test sample comprising one or more test compounds, and the test system comprising a system for evaluating half-channel blockade, the system being characterized by exhibiting elevated transfer of, for example, dyes or labeled metabolites, in response to the introduction of hyperglycemia, hypoxia or ischemia into the system, inflammatory mediators, or other compounds or events that induce half-channel opening, such as extracellular Ca2+. 2+ (a) Determine the decrease in the concentration of the metabolite; and (b) determine the presence or increase in the amount of a metabolite, such as a dye or other labeled metabolite, in the system. Positive and / or negative controls may also be used. Optionally, a predetermined amount of a half-channel blocker (e.g., peptide 5 or Xiflam) may be added to the test system.

[0206] Dosage form, formulation and application

[0207] Unless otherwise explicitly stated, all descriptions regarding the dosage of the drug apply to the half-channel blockers of this invention.

[0208] The semi-channel blocker can be administered, applied, or formulated as described in this article.

[0209] In one embodiment, a combination comprising, substantially comprising, or composed of one or more hemichannel blockers is administered. Hemichannel blockers may be administered once daily, twice daily, three times daily, four times daily, or in weekly doses, such as once weekly (once a week) or twice weekly (twice a week). They may also be administered monthly using the doses described herein. They may also be administered as needed (i.e., as required) and at bedtime (i.e., before sleep).

[0210] The hemichannel blocker can be administered to the subject requiring treatment. Therefore, according to the invention, a formulation is provided that can modulate connexin hemichannels (e.g., connexin 43 hemichannel, connexin 45 hemichannel, or connexin 36 hemichannel) to reduce their probability of opening in a transient and site-specific manner.

[0211] The hemichannel blocker can be present in the formulation in a substantially separate form. It should be understood that the product can be mixed with a carrier or diluent that does not interfere with the intended purpose of the product and is still considered substantially separate. The product of the present invention can also be in a substantially purified form, in which case it typically contains, for example, about 80%, 85%, or 90% (e.g., at least about 88%, at least about 90%, 95%, or 98%, or at least about 99%) of a small molecule hemichannel blocker, or the dry matter of the formulation.

[0212] A semichannel blocker can be administered to a subject by any means capable of delivering the drug to a target site within the subject's body. As examples, a semichannel blocker can be administered via one of the following routes: oral, local, systemic (e.g., intravenous, intra-arterial, intraperitoneal, transdermal, intranasal, or via suppository), parenteral (e.g., intramuscular, subcutaneous, intravenous, or intra-arterial injection), via implantation (including peritoneal, subcutaneous, and ocular implantation), and via infusion (via devices such as osmotic pumps, transdermal patches, etc.). Exemplary administration routes are also outlined in: Binghe, W., and B. Wang (2005). Drug delivery: principles and applications, Binghe Wang, Teruna Siahaan, Richard Soltero, Hoboken, NJ Wiley-Interscience, c2005. In one embodiment, the semichannel blocker is administered systemically. In another embodiment, the semichannel blocker is administered orally. In yet another embodiment, the semichannel blocker is applied, for example, locally to the eye or directly into the eye.

[0213] In some aspects, the hemichannel blocker may be provided as an implant or in combination with an implant. In some aspects, the implant may provide slow-release, controlled-release, or sustained-release delivery, with or without a burst dose. In some embodiments, a micromanipulation needle, needle tip, iontophoresis device, or implant may be used for the administration of the hemichannel blocker. The implant may be, for example, a soluble disc material, such as that described in S. Pflugfelder et al., ACS Nano, 9(2), pp. 1749-1758 (2015). In some aspects, the hemichannel blocker of the present invention, such as the connexin 43 hemichannel blocker, may be administered via intraventricular, and / or intrathecal, and / or epidural, and / or subdural, and / or supradural routes.

[0214] Semichannel blockers can be administered once, more than once, or periodically. They can also be administered as needed, according to a predetermined schedule, or both. In some respects, semichannel blockers are administered daily, weekly, monthly, bi-monthly, or quarterly, or any combination of these time periods. For example, treatment may be administered daily for a period of time, followed by weekly and / or monthly administration, and so on. This article characterizes other methods of administering the blocker. In one respect, a sufficient amount of semichannel blocker to treat the patient is administered at times 1 to 5, 10, 30, 45, 60, 75, 90 days, or 100 to 180 days, or between.

[0215] Half-channel blockers (such as compounds of formula I, such as Xiflam, and any analogues or prodrugs of the aforementioned compounds, or compounds of formula II) can be administered alone or in combination with one or more additional ingredients, and can be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable excipients, diluents, and / or carriers. In some embodiments, the half-channel blocker (such as compounds of formula I, such as Xiflam (tonaboxa), and any analogues or prodrugs of the aforementioned compounds, or compounds of formula II) can be administered orally in a composition comprising a food ingredient. In some embodiments, the food ingredient is peanut butter or hazelnut-based butter. Without being bound by theory, it is believed that relatively hydrophobic compounds of formula I, including tonaboxa, or compounds of formula II, are slowly released after being encapsulated in the emulsified fat of a food (e.g., peanut butter), thereby resulting in a prolonged therapeutic life.

[0216] As used herein, the term "pharmaceutically acceptable diluent, carrier, and / or excipient" is intended to include substances that can be used to prepare pharmaceutical compositions, can be co-administered with compounds of Formula I (e.g., Xiflam, and any analogues of the aforementioned compounds) or compounds of Formula II, while allowing them to perform their intended function, and are generally safe, non-toxic, and not biologically or otherwise undesirable. Pharmaceutically acceptable diluents, carriers, and / or excipients include those suitable for veterinary use as well as for human pharmaceutical use. Suitable carriers and / or excipients will be readily understood by those skilled in the art in light of the properties of compounds of Formula I (e.g., Xiflam, and any analogues of the aforementioned compounds). However, by way of example, diluents, carriers, and / or excipients include solutions, solvents, dispersion media, delay agents, polymers and lipid agents, emulsions, etc. As further examples, suitable liquid carriers (especially for injectable solutions) include water, saline solutions, glucose solutions, etc., isotonic solutions are preferably used for intravenous, intraspinal, and intracisional administration, and media such as liposomes are also particularly suitable for the administration of pharmaceutical preparations.

[0217] The composition can take the form of any standard known dosage form, including tablets, pills, capsules, semi-solids, powders, sustained-release formulations, solutions, suspensions, elixirs, aerosols, injectable liquids, gels, creams, transdermal delivery devices (e.g., transdermal patches), inserts such as organ inserts, such as those to the skin or eye, or any other suitable composition. Those skilled in the art to which this invention pertains will readily understand the most suitable dosage form considering the nature of the condition to be treated and the active agent to be used, without any excessive experimentation. It should be understood that one or more of the semi-channel blockers (such as compounds of formula I, such as Xiflam, and any analogues of the aforementioned compounds, and / or compounds of formula II) can be formulated into a single composition. In some embodiments, preferred dosage forms include injectable solutions, implants (preferably slow-release, controlled-release, or sustained-release implants, with or without burst-release doses), and oral formulations.

[0218] Considering dosage form and administration method, compositions used in this invention may contain any suitable level of a half-channel blocker, such as compounds of formula I, for example Xiflam, and any analogues of the aforementioned compounds, and / or compounds of formula II. However, as an example, compositions used in this invention may contain from about 0.1% to about 99%, preferably from about 1% to about 60% by weight of a half-channel blocker, depending on the method of administration.

[0219] In addition to standard diluents, carriers, and / or excipients, the compositions according to the invention can be formulated with one or more additional components, or formulated in a specific manner, for example, to enhance the activity or bioavailability of half-channel blockers (such as compounds of formula I, such as Xiflam, and any analogues of the aforementioned compounds, and / or compounds of formula II), to help protect their integrity or increase their half-life or shelf life, to enable slow release after administration to a subject, or to provide other desired benefits. For example, sustained-release mediators include macromonomers, poly(ethylene glycol), hyaluronic acid, poly(vinylpyrrolidone), or hydrogels. As further examples, the compositions may also include preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, coating agents, buffers, etc. Those skilled in the art to which this invention pertains can identify other additives that may be necessary for a particular purpose.

[0220] As noted, a semi-channel blocker can be administered via a sustained-release system. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of molded articles, such as films or microcapsules. Sustained-release matrices include polylactide (US Patent No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release compositions also include compounds encapsulated in liposomes. Liposomes containing hemichannel blockers can be prepared using known methods, including, for example, those described in the following documents: DE 3,218,121; EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese Patent Application 83-118008; US Patents 4,485,045 and 4,544,545; and EP102,324. Typically, liposomes are small (from or about 200 to 800 Å) monolayers containing a lipid content greater than about 30 mol% cholesterol, the selected proportions adjusted for the most effective treatment. For example, sustained-release delivery can also be used, employing PGLA nanoparticles or microparticles or in-situ ion-activated gelation systems.

[0221] Furthermore, it is anticipated that the hemichannel blocker pharmaceutical compositions used according to the present invention can be formulated with additional active ingredients or agents that, in certain circumstances, have therapeutic or other benefits for the subject. Suitable additional active ingredients will be understood by those skilled in the art, taking into account the description of the invention herein and the nature of the disorder to be treated.

[0222] Furthermore, the semi-channel blocker pharmaceutical composition used according to the present invention is intended to be formulated in candies or foods, for example, as a "gel-like" drug.

[0223] Compositions can be formulated according to standard techniques, as can be found in, for example, standard references such as Gennaro AR: Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams & Wilkins, 2000. However, as a further example, the information provided in US2013 / 0281524 or US5948811 can be used.

[0224] Any container suitable for storing and / or administering the pharmaceutical composition can be used for the semi-channel blocker product used in the method of the present invention.

[0225] Hemichannel blockers, such as connexin 43 hemichannel blockers, can, in some aspects, be formulated to provide controlled and / or compartmentalized release to the site of application. In some aspects of the invention, the formulation can be an immediate-release dosage form, or an extended-release dosage form, or a sustained-release dosage form. In some aspects, the dosage form can include an immediate-release dosage form in combination with an extended-release and / or sustained-release dosage form. In some aspects, by combining a hemichannel blocker in an immediate-release form, immediate and sustained and / or extended release of the hemichannel blocker can be obtained. In some aspects of the invention, the hemichannel blocker is, for example, a connexin 43 blocker or other hemichannel blockers of this disclosure. In some aspects of the invention, the dosage form can be an implant, for example, a biodegradable or non-biodegradable implant.

[0226] This invention includes methods for modulating the function of hemichannels to treat and reverse or substantially reverse or improve various disorders. The methods of this invention include administering hemichannel blockers, alone or in combination with one or more other agents or therapies, as needed.

[0227] The administration of a hemichannel blocker and optionally one or more other active agents can occur at any time during the progression of the disorder, or before or after the development of the disorder or one or more symptoms of the disorder. In one embodiment, the hemichannel blocker is administered periodically for an extended phase to aid in ongoing management or symptom reversal. In another embodiment, the hemichannel blocker is administered periodically during an extended phase or throughout life to prevent or delay the development of the disorder or to eliminate the disorder.

[0228] In some embodiments, the hemichannel blocker, such as a connexin 43 hemichannel blocker (e.g., compounds of formula (I), including tonabosa, or compounds of formula (II)), may be administered as a pharmaceutical composition comprising one or more particles. In some aspects, the pharmaceutical composition may be, for example, an immediately released formulation or a controlled-release formulation, such as a delayed-release particle. In other aspects, the hemichannel blocker may be formulated as a microparticle formulation, with one or more particles for selective delivery to a treatment area. In some embodiments, the particles may be, for example, nanoparticles, nanospheres, nanocapsules, liposomes, polymeric micelles, or dendritic polymers. In some embodiments, the particles may be microparticles. Nanoparticles or microparticles may comprise a biodegradable polymer. In other embodiments, the hemichannel blocker is prepared or administered as an implant or matrix, or formulated to provide septate release to the application site. In some embodiments, pharmaceutical compositions of hemichannel blockers, such as connexin 43 hemichannel blockers (e.g., compounds of formula (I), including tonabosa, or compounds of formula (II)), do not contain microparticles.

[0229] In some embodiments, as indicated, as an example, the formulated hemichannel blocker is a connexin 37, connexin 40, connexin 43, or connexin 45 hemichannel blocker. Connexin 36, connexin 37, connexin 40, connexin 43, or connexin 45 blockers are preferred. Connexin 36 and connexin 43 hemichannel blockers are most preferred. Connexin 43 hemichannel blocker is particularly preferred. The term "matrix" as used herein includes, for example, matrices such as polymer matrices, biodegradable or non-biodegradable matrices, and other carriers that can be used to manufacture implants or application structures for delivering hemichannel blockers. Implants include reservoir implants and biodegradable matrix implants.

[0230] Products / kits containing combinations of protein hemichannel blockers

[0231] In another embodiment of the invention, an article or "kit" is provided containing materials that can be used to treat the aforementioned diseases and disorders. The kit includes a container containing, substantially consisting of, or consisting of a hemichannel blocker. The kit may further include a label or instruction manual on or associated with the container. The term "instruction manual" is used to refer to the instructions typically included in the commercial packaging of a therapeutic product, containing information regarding indications, usage, dosage, administration, contraindications, and / or warnings for the use of such a therapeutic product. Suitable containers include, for example, bottles, tubular vials, syringes, blister packs, etc. Containers can be formed from a variety of materials such as glass or plastic. The container contains a hemichannel blocker or a formulation thereof that can effectively treat the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a bottle with a stopper that can be punctured by a hypodermic needle). The label or instruction manual indicates that the composition is used to treat selected conditions, such as any diseases, disorders, and / or conditions described or mentioned herein. The label or instruction manual may also indicate that the composition can be used to treat other disorders. Alternatively, or additionally, the finished product may further include a second container containing pharmaceutically acceptable buffer solutions, such as water for injection with antibacterial properties (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may further include other materials deemed commercially and user-appropriate, including other buffer solutions, diluents, filters, needles, and syringes.

[0232] The kit may further include instructions for administering a semi-channel blocker to patients who require it.

[0233] Articles are also provided that comprise, are substantially composed of, or are composed of: a container containing a hemichannel blocker compound, composition, or formulation, and instructions for use for treating a subject. For example, in another aspect, the invention includes an article comprising, is substantially composed of, or is composed of: a container containing a therapeutically effective amount of one or more connective protein hemichannel blockers, including small molecules, and instructions for use, including for treating a subject.

[0234] In some respects, the article may comprise a matrix containing one or more linker protein hemichannel blockers, either individually or in combination, such as small molecule hemichannel blockers.

[0235] Dosage, amount and concentration

[0236] As will be understood, the dosage, stage of administration, and general administration regimen of the administered hemichannel blocker may vary from subject to subject, depending on variables such as the target site to which it is delivered, the severity of any symptoms in the subject to be treated, the type of disorder to be treated, the size of the unit dose, the chosen administration modality, and the subject’s age, sex, and / or general health, as well as other factors known to those skilled in the art.

[0237] This article includes methods for increasing the survival of retinal structure and / or function and / or choroidal structure and / or function, and for salvaging or restoring retinal structure and / or function and / or choroidal structure and / or function in subjects with such need, including, for example, administering to the subject an effective amount of a hemichannel blocker, including, for example, N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrotryptene-4-yl]-3-chloro-4-fluorobenzamide (Xiflam). In some embodiments, the survival-promoting dose is about 10 to about 200 mg per day, or in some embodiments, about 3.5 to 350 mg per day. In other embodiments, the survival-promoting dose is about 20 to about 100 mg per day. These doses may be administered as a single dose or in divided doses, for example, twice daily. Preferred doses are in the range of about 0.5 to about 5 mg / kg per day. The dosage may be, for example, about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9 or about 5.0 mg / kg per day, or any range between any two of the stated dosages.

[0238] A particularly preferred daily dose is about 1.4 mg / kg per day, in a single dose or divided doses (e.g., twice daily). Thus, for example, for subjects weighing about 70 kg, 90 kg, or 100 kg, the daily dose would be about 98 mg, about 126 mg, or about 140 mg, respectively. These doses will provide an effective peak steady-state concentration of the half-channel blocker (e.g., Xiflam) after approximately 10 days.

[0239] Importantly, for efficacy and for patient convenience and compliance, other dosages and useful weekly, monthly, and implant dosing and dosing regimens have also been discovered and are provided herein. Some preferred weekly dose ranges are from about 2 mg / kg to about 50 mg / kg. Weekly doses can be, for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or about 50 mg / kg, or any range between any two of the stated weekly doses. For example, once-weekly doses of about 42 to about 47 mg / kg provide an effective trough half-channel concentration for a half-channel blocker (e.g., a half-channel blocker of formula I or II, such as Xiflam) with an expected half-life of about 4-5 per week, for implementing the methods of the present invention concerning the structure and function of the retina and / or choroid. Peak plasma concentrations at doses of about 42 to about 47 mg / kg will be higher than effective trough concentrations, but are tolerable. When administered monthly, doses of 25-100 mg / kg will also be effective.

[0240] In some embodiments, the survival-promoting dose is about 4.5 to about 450 mg administered once weekly. These doses include doses from about 4.5 to about 45 mg once weekly and doses from about 45 to 450 mg once weekly, or any dose in between. A dose obtained by multiplying any weekly dose disclosed herein by the patient's weight (e.g., 60, 65, 70, 75, 80, 85, 90, 95, or 100 kg) may also be used.

[0241] In another weekly (once-a-week) dosing regimen, the hemichannel blocker compound is administered in the form of a slow-release, sustained-release, or controlled-release oral or implantable formulation, with or without a 10-20% burst dose or other desired burst dose. Implantable formulations, such as ocular implants, are preferably formulated as slow-release, sustained-release, or controlled-release oral or implantable formulations.

[0242] Doses of 3.5 to 350 mg daily, 10 to 200 mg daily, or 20 to 100 mg daily can also be used to salvage or restore retinal structure and / or function, and to salvage or restore choroidal structure and / or function. In some embodiments, an oral dose of 15-150 mg, 25-250 mg, 40-400 mg, or 80-800 mg of the anti-hemispheric compound is administered as a single or fractionated dose as an amount to promote the survival of retinal and / or choroidal function, to salvage or restore retinal structure and / or function, or to salvage or restore choroidal structure and / or function. In other embodiments, an oral dose of 100-500 mg, 500-1000 mg, or 1000-2000 mg is administered as a single or fractionated dose. Fractionated doses may be administered twice daily, three times daily, four times daily, or once weekly. Xiflam is currently the preferred compound for oral administration in measured doses.

[0243] Importantly, weekly doses can be used to salvage or restore retinal structure and / or function, or choroidal structure and / or function. Importantly, higher doses, such as 500 mg to 2000 mg, or amounts between these doses, such as 750 mg, 1000 mg, 1250 mg, 1500 mg, and 1750 mg, need to be administered only once weekly or even once monthly for the purpose of salvaging or restoring retinal structure and / or function, or choroidal structure and / or function. Xiflam is currently the preferred compound for oral administration in these amounts. Other weekly doses include doses from about 2500 to 5500 mg, with preferred doses equal to about 2900 mg, 3700 mg, 4200 mg, 3300 mg, 4200 mg, and 4700 mg once weekly, and all doses in between. These doses are also effective when administered monthly.

[0244] Examples of effective doses that can be used to treat the diseases, disorders, or conditions mentioned herein are described. Other exemplary doses are in the range of about 0.1 to about 5.0 mg / kg, including, for example, 0.2 to 3.0 mg / kg, or 0.2 to 2 mg / kg and 0.2 to 1.0 mg / kg, or 0.2 to 0.5 mg / kg. Some exemplary daily or other periodic dose ranges are about 10-250 mg per dose, including, for example, about 20-25 mg per dose, about 25-50 mg per dose, about 20-40 mg per dose, about 50-75 mg per dose, about 75-100 mg per dose, and about 100-250 mg per dose, including doses of 20, 50, 100, and 150 mg per dose, or any specific dose falling within these mg drug / kg body weight ranges. In some embodiments, the circulating concentrations of the half-channel blocker (including compounds of formula (I), including tonabosar, and compounds of formula (II)) in subjects receiving the half-channel blocker range from about 5 μmol to about 200 μmol, from about 7 μmol to about 100 μmol, or from about 10 μmol to about 90 μmol.

[0245] As noted above, the dose of a hemichannel blocker (e.g., a hemichannel blocker of connexin 37, 40, or 43) may be administered in a single or separate application. The dose may be administered once or repeatedly. Typically, it may be repeated weekly, every two or three weeks, monthly, or every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 months, or every 24 months or longer, as needed, to prevent, alleviate, or treat any of the diseases, disorders, or conditions described herein. The dose may also be administered at intervals of 12 hours to 7 days or longer. For example, the dose may be administered at intervals of 12 hours or 1, 2, 3, 4, 5, 6, or 7 days, or at any time interval falling between any two of these times, or between 12 hours and 7 days. For example, connexin 43 hemichannel blockers can be administered for up to four, six, eight, ten, twelve, fourteen, sixteen, eighteen, twenty, twenty-two, twenty-four, or twenty-six weeks. For certain indications, more frequent dosing may be used. In some embodiments, the hemichannel blocker may be administered daily at an initial dose level for a first period, and then daily at an increasing dose level for another period.

[0246] Preparation and purity

[0247] Small molecule half-channel blockers, including those of formulas I and II, can be prepared as described above.

[0248] In some embodiments, the formulations of the present invention are substantially pure. Substantially pure means that the formulation contains less than about 10%, 5%, or 1%, and preferably less than about 0.1%, of any impurities. In some embodiments, the total impurities, including metabolites of the connexin 43 regulator, will not exceed 1-15%. In some embodiments, the total impurities, including metabolites of the connexin 43 regulator, will not exceed 2-12%. In some embodiments, the total impurities, including metabolites of the connexin 43 regulator, will not exceed 3-11%. In other embodiments, the total impurities, including metabolites of the connexin 43 regulator, will not exceed 4-10%.

[0249] Example

[0250] The work described in these embodiments evaluates and demonstrates the ability of treatment with hemichannel blocker doses and dosing regimens to maintain choroidal thickness, retinal thickness, and preserve and salvage retinal function in animals with diabetic retinopathy.

[0251] Example 1

[0252] method

[0253] A model of retinal light damage in age-related macular degeneration – the strong light damage model was prepared and implemented as described in previous studies. Mat Nor N, Guo CX, Rupenthal ID, Chen YS, Green CR, Acosta ML. SustainedConnexin43 Mimetic Peptide Release From Loaded Nanoparticles Reduces Retinaland Choroidal Photodamage. Invest Ophthalmol Vis Sci. 2018;59:3682-93; Guo CX, Mat Nor MN, Danesh-Meyer HV, Vessey KA, Fletcher EL, O'Carroll SJ, et al. Connexin43Mimetic Peptide Improves Retinal Function and Reduces Inflammation in a Light-Damaged Albino Rat Model. Invest Ophthalmol Vis Sci. 2016;57:3961-73; GuoCX, Tran H, Green CR, Danesh-Meyer HV, Acosta ML. Gap proteins junction in thelight-damaged albino rat.Mol Vis. 2014; 20: 670-82; Noell WK, Walker VS, Kang BS, Berman S. Retinal damage by light in rats. Invest Ophthalmol. 1966; 5: 450-73. The photodamage rat model was chosen because it allows for direct comparison of this drug with other drugs.Kim Y, Griffin JM, Nor MNM, Zhang J, Freestone PS, Danesh-Meyer HV, et al. Tonabersat Prevents Inflammatory Damage in the Central Nervous System by Blocking Connexin43 Hemichannels. Neurootherapeutics. 2017;14:1148-65; Mat Nor N, Guo CX, Rupenthal ID, Chen YS, Green CR, Acosta ML, ibid.; Guo CX, Mat Nor MN, Danesh-Meyer HV, Vessey KA, Fletcher EL, O′Carroll SJ, et al., ibid. The model confirms the pathological factors of AMD (oxidative stress and inflammation) and allows for measurable endpoints (including retinal electrical function). A drawback of this model is that, as with other rodent models, drusen does not develop. All experimental procedures were approved by the University of Auckland Animal Ethics Committee, approval number 001462, and in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement on the use of animals in ophthalmic research. Six- to eight-week-old albino Sprague Dawley (SD) rats (200–250 g; male or female) were used. Adult SD rats were exposed to continuous, strong light for 24 hours, consistently starting at 9:00 AM to minimize possible temporal variability throughout the day. Two animals were exposed to light at a time to prevent rats from using each other as shields. The LD protocol and intervention were repeated until the number of individuals in each dose group (n = 7 per group) was obtained. The light intensity was 2700 lux, generated directly above the animal cages using a fluorescent lamp (Philips Master TLD18W / 965; Koninklijke Philips Electronics NV, China). The lamp was cold and emitted broadband light at wavelengths from 380 to 760 nm, with an average intensity of 120 W / m² at the top of the cages. Animals were allowed free movement within their cages and access to food and water at will. Baseline electroretinography (ERG) readings and optical coherence tomography (OCT) images were collected prior to light exposure. After light exposure, animals were returned to normal light-dark cycles (12 hours of light, 174 lux, and 12 hours of darkness, <62 lux) for 24 hours, 1 week, or 2 weeks (with one set lasting 3 months).

[0254] Tonabosa Treatment in Photodamaged Rats – LD rats were randomly assigned to low, medium, or high doses of tonabosa (n=7 per group). A fresh tonabosa mixture was prepared in peanut butter for each experiment. Three oral doses of tonabosa were tested (n=7 per group), with follow-up up to 2 weeks post-injury. The highest-dose group was then divided into three animals for histological analysis, and four animals were followed up up to 3 months post-injury. A ten-animal, vehicle-only control group was present. Rats fed for 3 months were maintained in separate cages as animals treated with either the drug or the vehicle, but under the same light conditions. Tonabosa was administered to animals in peanut butter at 0.26 mg / kg (mean delivery 0.08 mg, estimated circulating concentration 10 μM), 0.8 mg / kg (mean delivery 0.24 mg; 30 μM circulating), or 2.4 mg / kg (mean delivery 0.72 mg; 90 μM circulating), after accounting for previous unsuccessful human trials. Silberstein SD. Tonabersat, a novel gap-junction modulator for the prevention of migraine. Cephalalgia. 2009; 29 Suppl 2: 28-35; Dahlof CG, Hauge AW, Olesen J. Efficacy and safety of tonabersat, a gap-junction modulator, in the acute treatment of migraine: a double-blind, parallel-group, randomized study. Cephalalgia. 2009; 29 Suppl 2: 7-16; Goadsby PJ, Ferrari MD, Csanyi A, Olesen J, Mills JG, Tonabersat TONSG. Randomized, double-blind, placebo-controlled, proof-of-concept study of the cortical depression spreadinginhibiting agent tonabersat in migraine prophylaxis.Cephalalgia.2009;29:742-50. Animals were fed immediately before the light exposure phase; those not treated with drugs were excluded from the study (total experiment, n = 29 animals treated with drugs and vectors). The treatment groups were known to the researchers at the time of treatment but were subsequently randomized before statistical comparison, meaning that the analysis was conducted without knowing which animals were treatment or control.In summary, adult rats were acclimatized to darkness overnight and ERG data were collected. Animals were fed a medium or tonabothia prior to exposure to strong light. Two weeks later, animals were again evaluated by ERG and OCT, and tissues were collected immediately thereafter, except for four rats in the high-dose group who underwent an additional 3-month experiment.

[0255] A hyperglycemic rat model of diabetic retinopathy – A spontaneously hyperglycemic strain of SD rats was identified and isolated at the Vernon Jansen Animal Research Unit, Faculty of Medicine and Health Sciences, University of Auckland, exhibiting clinical signs of diabetic retinopathy within 4 weeks of birth. Although precise information about the etiology of the disease is lacking, the identification of these rats exhibiting hyperglycemia and microaneurysms provides an opportunity to treat complex chronic disease models and assess treatment efficacy based on objective, measurable endpoints. Inbreeding was performed for three generations, and ocular abnormalities were screened between 4 and 8 weeks of age. Further information about these rats is provided as supplementary information. Glucose levels were tested in non-fasting rats using a Freestyle Optium blood glucose meter (Abbott Laboratories Ltd., UK) and Freestyle Optium glucose test strips (Lee JJ, Yi HY, Yang JW, Shin JS, Kwon JH, Kim CW. Characterization of streptozotocin-induced diabetic rats and pharmacodynamics of insulin formulations. Biosci Biotechnol Biochem. 2003; 67: 2396-401).

[0256] Ten rats (10 normal SD rats and 10 hyperglycemic rats) were selected and grown to 5 weeks of age, and evaluated using OCT and ERG. The hyperglycemic rats were then divided into two subgroups of five rats each. One subgroup was fed tonaboxacillin at a low dose of 0.28 mg / kg in 0.5 g peanut butter once daily for 14 days from week 5 to week 7, while the other subgroup was fed 0.5 g peanut butter alone. All animals were again evaluated using ERG and OCT at 8 weeks of age, and then euthanized with enucleation for immunohistochemical analysis. Data sets were randomized before statistical comparison. In summary, normal SD and hyperglycemic rats were analyzed using OCT and ERG at 5 weeks of age; the hyperglycemic rats were then divided into two groups and fed either the tonaboxacillin or the carrier for 14 days during weeks 6 and 7. At week 8, the animals were again evaluated using ERG and OCT, and tissues were collected for immunohistochemical analysis. However, four tonaboza-treated rats were left until they were 3 months old before the final ERG and OCT and tissue collection.

[0257] Evans Blue Dye Assessment of Vascular Leakage – To investigate whether microaneurysms and aortic aneurysms in the retina of hyperglycemic rats (observed using OCT) reflect the site of vascular leakage, rats were perfused with Evans blue dye at 3 months of age, as previously described. Cai S, Yang Q, Hou M, Han Q, Zhang H, Wang J, et al. Alpha-Melanocyte-Stimulating Hormone Protects Early Diabetic Retina from Blood-Retinal Barrier Breakdown and Vascular Leakage via MC4R. Cell Physiol Biochem. 2018;45:505-22. Briefly, Evans blue dye (30 mg / ml; Sigma-Aldrich, USA) was dissolved in physiological saline and filtered. The dye was delivered at 45 mg / kg as an injection into the tail vein of normal SD and hyperglycemic rats and allowed to circulate for 2 hours. The eyes were enucleated while the rats were deeply anesthetized, and the animals were euthanized by a rapid intragastric injection of 3M KCl. The posterior cup was fixed in 4% paraformaldehyde for 30 minutes, the retina was removed and placed flat. Evans blue was applied at a wavelength of 559 nm and visualized by its red fluorescence emission using an Olympus FluoView FV1000 (Olympus Corporation, Tokyo, Japan).

[0258] Electroretinography (ERG) recording – the procedure was performed as described previously. Vessey KA, Wilkinson-Berka JL, Fletcher EL. Characterization of retinal function and glial cell response in amouse model of oxygen-induced retinopathy. J Comp Neurol. 2011; 519: 506-27. Essentially, SD rats underwent overnight dark acclimatization for 12–14 hours before ERG recording. For dry AMD, baseline ERG was recorded for all groups at time points before and after light exposure (24 hours, 1 week, 2 weeks, and 3 months after intense light exposure). For the DR model, ERG was recorded at 5 weeks of age to compare retinal function in normal SD and hyperglycemic rats. Tonaboxa-treated and mediator-controlled hyperglycemic rats were reassessed at 8 weeks of age. After dark acclimatization, rats were anesthetized by a combination of intraperitoneal injection of ketamine (75 mg / kg, Parnell Technologies, New Zealand) and domitor (0.5 mg / kg, Pfizer, New Zealand). Dark-adapted animals were operated on with dark red light (λmax = 650 nm) generated by LEDs. During ERG recording, the cornea was kept hydrated with 1% sodium carboxymethyl cellulose (Celluvisc, Allergan, USA). ERG was recorded in both the right and left eyes using gold ring electrodes (Roland Consult Stasche and Finger GmbH, Germany). U-shaped active electrodes were kept in contact with the center of the cornea. V-shaped inactive electrodes were hooked around the anterior teeth and in contact with the moist tongue. Normal body temperature was maintained by placing the animals on a 37°C heating pad to avoid temperature-driven fluctuations in ERG amplitude. A full-field ERG response was elicited via a double flash (0.8 ms stimulus interval) generated by a photographic flash unit (Nikon SB900 flash, Japan) through a Ganzfeld sphere. An integrating sphere, approximately 650 mm in diameter and coated with white, was used to reflect the flash onto the entire retina. Flash intensities ranged from -2.9 to 2.1 log cd.s / m², and were attenuated using a neutral density filter (Kodak Wratten, Eastman Kodak, USA) to obtain light intensities of -3.9, -2.9, -1.9, 0.1, 1.1, 1.6, 1.8, and 2.1 log cd.s / m². Flash intensities were calibrated using an IL1700 research radiometer (UV ProcessSupply Inc., USA). This study utilized a dual-flash mode to isolate the rod and cone paths. Pairs of flashes with identical light energy were triggered from the flash unit.After the first flash, a mixed response of rod and cone was recorded, and the response of the second flash was recorded, representing only the function of the cone. The rod PIII response was obtained by subtracting the cone response from the initial mixed response. The PIII component of the ERG is a direct reflection of the rod photocurrent, and by fitting its response to a computational model, the slope of the a-wave can be more appropriately explained by taking into account the photocurrent information of the rod. To this end, the ERG data at the highest illumination level were fitted to the rod response model, assuming an initial linear rise and then saturation of the response amplitude and intensity, to show the PII (bipolar cellular component) and PIII (photoreceptor component). Through this separation of rod PII and PIII, we can confirm that the a-wave and b-wave ERG data correspond to changes in the cone and rod pathways. Oscillatory potential (OP) is another method for studying inner retinal function. OP is separated by subtracting the original b-wave from the rod PII. Weymouth AE, Vingys AJ. Rodent electroretinography: methods for extraction and interpretation of rod and cone responses. Prog Retin Eye Res. 2008; 27: 1-44. The total amplitudes of OPs 2, 3, and 4 were analyzed. Recording was performed in a Faraday cage to reduce electrical noise. The results of the ERG signals were amplified 1,000 times using a Dual Bio Amp (AD Instruments, Australia), and the waveforms were recorded using Scope software (AD Instruments, New Zealand) and analyzed using publicly available algorithms for the a-wave and b-wave amplitudes of each eye. Guo CX, Mat Nor MN, Danesh-Meyer HV, Vessey KA, Fletcher EL, O'Carroll SJ, et al., ibid.; Vessey KA, Wilkinson-Berka JL, Fletcher EL, ibid. To achieve 80% power and 5% α value, we determined that a sample size of 5 was required for the ERG studies.

[0259] Optical coherence tomography—Spectral domain optical coherence tomography (SD-OCT; Micron IV; Phoenix Research Laboratories, USA) was used to obtain information on the morphology of the retinal layers in vivo. OCT was performed immediately after ERG recording, with animals anesthetized and pupils dilated using 1% tropicamide (Bausch & Lomb New Zealand Ltd., New Zealand). Guo CX, Mat Nor MN, Danesh-Meyer HV, Vessey KA, Fletcher EL, O'Carroll SJ, et al., ibid. Rats were placed on a 37°C heating pad to maintain body temperature and prevent cold cataracts. The eyes were covered with Poly Gel (containing 3 mg / g carbomer; Alcon Laboratories Pty Ltd, Australia), and the retina was imaged by contacting the OCT lens with the gel. StreamPix 6 software version 7.2.4.2 (Phoenix Research Laboratories, USA) was used for image acquisition. SD-OCT horizontal B-scans with 2 μm axial resolution were performed, with each A-scan consisting of 1024 pixels. Ten B-scans, each 2 mm long, were acquired from the optic nerve in the dorsal retina and averaged. Images were analyzed using InSight software version 1.1.5207 (Phoenix Research Laboratories, USA). Choroidal layer thickness was measured from the highly reflective Bruch's membrane to the choroid-scleral interface. Outer nuclear layer (ONL) thickness was measured from the outermost membrane (OLM) to the outermost plexiform layer (OPL) interface.

[0260] Tissue Collection and Processing – At the end of the final OCT recording, rats were deeply anesthetized using a combination of ketamine (75 mg / kg, Parnell Technologies, New Zealand) and domitor (0.5 mg / kg, Pfizer, New Zealand). Animals were perfused with saline via the cervical canal for 2–3 minutes, followed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 (PB) for 30 minutes. The eye was dissected from the orbit, and the optic cups were further fixed by immersion in 4% paraformaldehyde, followed by washing in PB after 30 minutes. The tissues were then cryoprotected by passing through 10% and 20% sucrose / PB solutions for 30 minutes each at room temperature, followed by immersion overnight in 30% sucrose / PB at 4°C. The tissues were then embedded in an optimal cutting temperature compound (Sakura Finetek, Torrance, USA) and cryosectioned vertically (16 μm section thickness) using a Leica CM3050 S cryostat (Leica, Germany). Sections were collected on Superfrost Plus slides (Labserv, New Zealand) for immunohistochemical labeling. For DR animals, spleen, pancreas, liver, heart, and kidney were collected from randomly selected animals that had been injected with the vector (see Supplementary Information).

[0261] Immunohistochemical labeling of tissue sections – Frozen tissue sections were air-dried at room temperature for 10–15 minutes and washed with 0.1 M PB. Sections were surrounded with a PAP pen (Invitrogen, New Zealand) to form incubation wells and blocked at room temperature for 1 hour with 6% normal goat serum or donkey serum (Invitrogen, USA), 1% bovine serum albumin (BSA), and 0.5% Triton X-100 in 0.1 M PB. Primary antibodies included rabbit anti-connector 43 (1:1000, Cat C6219, Sigma-Aldrich, USA), mouse anti-Iba-1 (ionized calcium-binding connector molecule 1, 1:250, Cat Ab5076, Abcam, USA) specifically expressed by microglia, and mouse anti-GFAP (1:1000, Cat C9205, Sigma-Aldrich, USA) antibody for astrocytes and activated Müller cells. The slides were incubated overnight at room temperature with the primary antibody, then washed four times in 0.1M PB for 15 minutes each time. The secondary antibody (goat anti-rabbit or goat anti-mouse conjugated with Alexa™ 488 or Alexa™ 594 (Invitrogen, Australia)) was diluted 1:500 and applied in the dark at room temperature for 2–3 hours. The slides were then thoroughly washed with 0.1M PB, and the cell nuclei were stained with DAPI (1:1000; Sigma-Aldrich, USA), followed by coverslips with anti-fading medium (Citifluor Ltd, UK). The coverslips were sealed with nail polish. The slides were imaged using an Olympus FluoView FV1000 confocal laser scanning microscope (Olympus Corporation, Japan) equipped with excitation lasers at wavelengths of 405, 473, and 559 nm.

[0262] Graphing and statistical analysis were performed using GraphPad Prism 5 (GraphPad software, USA). All data are presented as mean ± standard error of mean (SEM). Analysis of variance (ANOVA) was used to compare functional and morphological data, with an α value of 0.05. Two-way ANOVA and subsequent Bonferroni post-hoc tests were used to compare the effects of stimulus intensity in the ERG response analysis. One-way ANOVA and subsequent Tukey's test were used in the ERG response at an intensity of 2.1 log cd.s / m² in controls and photodamaged animals, as well as in the OCT data analysis. Assuming a normal distribution of means among samples, unpaired t-tests with Welch correction were used for statistical analysis of the rod's PII and PIII.

[0263] Example 2

[0264] Treatment with hemichannel blockers preserved choroidal thickness, retinal thickness, and salvaged retinal function in an animal model of retinal degeneration caused by bright light damage.

[0265] The mixed a-wave ERG data plotted for the light intensity range of the experiment resulted in a negative deflection, increasing the flash intensity from low to medium range levels. The mixed b-wave ERG response was a positive deflection and was consistent for most flash intensities.

[0266] Twenty-four hours after light exposure, the ERG response in albino rats was significantly weakened, with a maximum a-wave amplitude of -100 μV. Figure 1 shows the ERG data for the animal group fed with the medium 2 weeks after injury and for each of the three treatment doses at 24 hours, 1 week, and 2 weeks after light injury.

[0267] At 24 hours after light exposure, there was no difference between the medium control group and any of the three tonaboxa dose groups (Figure 1).

[0268] However, a significant improvement in the mixed α-wave amplitude was observed in animals treated with 0.26 mg / kg and 0.8 mg / kg at 1 week post-treatment compared to the photodamaged control group (p < 0.01). Figure 1B -C), and in a wider range of intensity: 0.1–2.1 logcd.s / m2, in the 2.4 mg / kg treatment group (p < 0.001); Figure 1D ).

[0269] By 2 weeks post-treatment, all three doses of oral tonaboxa hemichannel blockers produced significant recovery of mixed alpha wave amplitude in intensities ranging from 0.1 to 2.1 log cd.s / m² (p < 0.001). Figure 1B -D). These animals exhibited an ERG α-wavelength at the highest intensity compared to the photodamage group treated with the medium (p < 0.001). Figure 1B -D), treatment resulted in the restoration of ERG function, only slightly below the average of approximately -600 μV in SD rats. In other words, all three doses of the hemichannel blocker restored ERG function.

[0270] At 24 hours post-treatment, the mixed b-wave function was significantly improved in the 0.26 and 0.8 mg / kg tonaboxacin treatment groups. Figure 1E -F), not observed in the high-dose treatment group ( Figure 1G However, for all three doses of tonaboxa, the mixed beta-wave amplitude increased at 1 and 2 weeks post-treatment, indicating a significant improvement in inner retinal function. Figure 1E-G). The highest dose of tonaboxax at 2.4 mg / kg showed the greatest improvement (mean 1200 μV), which was within the normal range of absolute amplitude of the b-wave in undamaged SD rats

[36] . For 0.26 and 0.8 mg / kg, there was improvement in absolute amplitude of the b-wave (mean 1000 μV). Nevertheless, at the end of the 2-week recovery period, the mixed a-wave and mixed b-wave functions of all tonaboxax-treated animals were within the range of variation of normal, undamaged albino rats. Heiduschka P, Schraermeyer U., Comparison of visual function in pigmented and albino rat by electroretinography and visual evoked potentials. Graefes Arch Clin Exp Ophthalmol. 2008;246:1559-73. In other words, all three doses of the hemichannel blocker salvaged inner retinal function.

[0271] Under normal reproductive and food access conditions, a group of four animals treated with the highest dose of oral tonabosa (2.4 mg / kg) maintained the treatment for 3 months. The benefits of oral tonabosa treatment were long-lasting. The ERG a-wave and b-wave wavelength amplitudes of the original seven treated animals, assessed 2 weeks after oral tonabosa (2.4 mg / kg), were only slightly lower than those of the group of four treated animals assessed 3 months after oral tonabosa. In the a-wave, the improvement in photoreceptor function exceeded 400 μV, and in the b-wave, the amplitude exceeded 800 μV compared to the control treated with the mediator. Figure 2A -B). In the PIII and PII rod responses, there were no changes after 3 months of oral tonaboxa treatment compared to before photodamage, indicating that tonaboxa treatment fully preserved photoreceptor function. In contrast, the photodamage group treated with only the medium showed significantly reduced changes in PIII and dPII rod amplitudes at 3 months (p < 0.001). Figure 2C -D). In other words, all three doses of the semichannel blocker salvaged photoreceptor function, as well as PIII rod and PII rod responses.

[0272] OCT scans were used to analyze the thickness of the retinal layer and choroid at 24 hours, 1 week, and 2 weeks post-injury. Figure 3 shows the typical appearance of the basal layer and OCT scans of normal adult Sprague Dawley rats, photo-injured rats treated with the mediator, and animals treated with 2.4 mg / kg tonaboxavar at 2 weeks post-injury. Figure 3A-C). Compared with the same eye before light damage, at the 2-week time point, the retina and choroid of the mediator-treated animals were significantly thinner (p < 0.001); Figure 3A -B). The loss of retinal thickness was primarily due to thinning of the ONL. However, all three doses of oral tonaboxa significantly preserved retinal and choroidal thickness, with no thinning detected at any of the treatment time points studied (24 hours, 1 week, and 2 weeks). Figure 3D -F). OCT analysis at 3 months after oral administration of tonaboxacin at 2.4 mg / kg showed that retinal and choroidal thickness were significantly preserved compared with the mediator treatment group (p < 0.001); Figure 4A -B). Medication-mediated treatment of the nuclear layer (INL) of animals; Figure 4C ) and ONL ( Figure 4D Thinning of the retina in the retinal layer was evident, but there was no difference in ONL thickness between the animals treated with oral tonabosa and the same animals before the light-damage procedure. The INL thickness in the oral tonabosa treatment group was slightly reduced at 3 months compared to the same animals imaged before the light-damage procedure (p < 0.05). The mediator-treated rats showed significant INL thinning compared to the same retinas before the light-damage procedure (p < 0.001). At 3 months, there was no difference in choroidal thickness between the oral tonabosa treatment rats and the same rats assessed before the light-damage procedure. Figure 4E In contrast, photodamaged rats treated with the mediator and assessed 3 months post-injury showed significant thinning of the choroid (p < 0.001). In other words, all three doses of the hemichannel blocker salvaged retinal and choroidal structures.

[0273] Following final ERG and OCT assessments, the posterior segment of the eye containing the retina and attached RPE-choroid-sclera was evaluated using immunohistochemical labeling with GFAP to investigate the extent of gliosis (astrocytosis), and Iba-1 was investigated to determine microglial cell immunoreactivity before and after treatment, as well as connexin 43. Rats treated with tonaboxa showed less connexin 43 immunoreactivity in the retina at all three tonaboxa doses used compared to those treated with the drug (Fig. 5A) (Fig. 5B-D). In the drug-treated group (Fig. 5F-H), the activity of Iba-1 immunolabeled cells in the inner plexiform layer (IPL) of the retina was lower than in the drug-treated group (Fig. 5E). Slightly higher levels of Iba-1 responsiveness were observed in rats treated with 0.26 mg / kg. Compared with rats treated with the medium (Fig. 5I), GFAP immunoreactivity was significantly higher at 0.8 mg / kg tonaboxa (Fig. 5K) and 2.4 mg / kg tonaboxa (Fig. 5K). Figure 5LNo increase was observed in the retina of the animals treated with tonaboxacin. In animals administered 0.26 mg / kg, GFAP markers were slightly increased (Fig. 5J), significantly lower than in rats treated with the carboxymethyl ether. Image quantification showed significantly reduced levels of GFAP, connexin 43, and Iba-1 in all tonaboxacin treatment groups compared to the carboxymethyl ether control group (p < 0.001). Figure 6A -C), exhibiting a dose-response trend (higher doses are more effective at maintaining normal levels of these retinal inflammatory markers).

[0274] Example 3

[0275] Treatment with hemichannel blockers salvaged retinal function in hyperglycemic animals with diabetic retinopathy.

[0276] The mean body weight of control SD rats was 185±1.1 g at 4 weeks of age, 198.2±0.8 g at 6 weeks of age, and 217.5±1.3 g at 8 weeks of age. Hyperglycemic rats had a lighter body weight, with mean body weights of 172.5±2.5 g at 4 weeks of age, 179.6±2.1 g at 6 weeks of age, and 183.1±1.8 g at 8 weeks of age. The differences between all three age groups and age-matched normal SD rats were statistically significant (t-test, p < 0.001). The blood glucose levels in normal SD rats ranged from 4.9 to 7.4 mmol / L (mean 6.07 mmol / L, with no significant difference between age groups); the glucose levels in hyperglycemic rats ranged from 14.0 to 21.0 mmol / L, with mean values ​​of 16.85 ± 0.63 mmol / L at 4 weeks, 15.43 ± 0.79 mmol / L at 6 weeks, and 16.54 ± 0.65 mmol / L at 8 weeks, maintaining a consistent hyperglycemic level from early age. The differences between hyperglycemic rats and normal SD rats in all three age groups were statistically significant (t-test, p < 0.001).

[0277] Slit-lamp examination of the anterior segment (cornea, lens) of 4-week-old hyperglycemic rats revealed no significant macroscopic differences compared to SD rats from which this strain was derived. The cornea, lens, and iris appeared identical to those of normal SD rats, with no signs of diabetic cataracts or neovascularization. However, OCT in the hyperglycemic rats showed an average of 5-8 hyperreflective spots per eye (based on 7 evenly distributed OCT scans of the retina, thus underestimating the entire eye), compared to no more than 1-2 in normal SD rats. These hyperreflective spots appeared to be microaneurysms (20-30 μm in diameter) and large aneurysms (140-160 μm). Figure 7B-C), which are specifically located in the INL and ONL. Although the choroidal thickness appeared to be slightly reduced in animals with aneurysms, there was no significant change in retinal or choroidal thickness. Evans blue dye perfusion confirmed vascular leakage at the aneurysm site mapped using OCT. Figure 7F ).

[0278] To determine whether aneurysms affect retinal function, ERG analysis was performed at 5 weeks of age to compare retinal function in hyperglycemic rats with that in normal SD rats. Representative ERG waveforms are shown in Figure 8. At intensities of 0.1–2.1 log cd.s / m², the mean mixed a-wave amplitude was significantly reduced in hyperglycemic rats compared to normal SD rats (p < 0.01), with SD rats measuring a maximum intensity of -630 μV, contrasting with -370 μV in the retina of hyperglycemic diabetic rats. For intensities of -3.9–2.1 log cd.s / m², the mixed ERG b-wave amplitude was also significantly reduced in hyperglycemic rats (p < 0.001), with normal SD rats exhibiting a maximum intensity of 800 μV, but only 400 μV in diabetic animals. No difference in implied time was observed between a-wave and b-wave. Further analysis showed that the amplitudes of rod PIII (p < 0.001), PII (p < 0.001), and cone PII (p < 0.001) responses, as well as the amplitude of the sum of oscillatory potentials (OP) (p < 0.001) in hyperglycemic rats were significantly reduced (Figure 8). No significant changes in rod PIII sensitivity or in rod PII, cone PII, and the sum of OP implied time.

[0279] For the tonaboxa DR treatment group, ten hyperglycemic rats reaching week 5 were divided into two equal groups. One group was fed tonaboxa at a low dose of 0.28 mg / kg once daily for 14 days from week 5 to week 7. At week 8, there was no difference in body weight, retinal layer thickness (INL or ONL), or choroidal thickness between the treated and untreated hyperglycemic rats. However, there were differences in the number and size of microaneurysms after treatment. Figure 9A -B). Compared with hyperglycemic rats injected with the medium, drug-treated hyperglycemic rats showed significant recovery of retinal function. Figure 9C Compared to -370 μV in untreated animals at 8 weeks, the intensity of the mixed ERG a-wave was significantly higher in treated animals in the range of 0.1–2.1 cd.s / m² (p < 0.001), with a maximum intensity of -630 μV, and closely matched the level in undamaged control SD rats. Figure 8A Similarly, in all animals treated with tonaboza, the mixed b-wave signal was significantly restored (p < 0.001), with a maximum intensity of 700 μV, compared to only 400 μV in the untreated control group at this time point. Figure 9D ), and then recovered to near-normal mixed b-wave values ​​in SD rats (see Figure 9B The implicit time was not different. Further analysis showed that hyperglycemic rats treated with the semi-channel blocker compound significantly restored the amplitudes of rod PIII (p < 0.001), PII (p < 0.001), cone PII (p < 0.01), and cumulative OP (p < 0.01). Figure 9E -H). There was no difference in the latency before and after treatment. In other words, the semi-channel blocker restored and salvaged the function and structure of the retina.

[0280] To determine whether the differences observed by OCT and ERG in hyperglycemic rats were related to retinal inflammation, the eyes were enucleated at 8 weeks for immunohistochemical examination. GFAP labeling was strong in the retinal ganglion cell (RGC) layer, and glial proliferation was observed in the perineurium of microaneurysms in the retina of hyperglycemic rats, extending from the nerve fiber layer (NFL) to the ONL, indicating Müller cell activation. Figure 10A Iba-1 markers are abnormally high in the hyperglycemic retina. Figure 10B This indicates that microglia in the inner retinal layer are activated, containing cells with enlarged cell bodies and numerous elongated branches, and that connexin 43 labeling in the GCL of hyperglycemic rats is abnormally high. Figure 10C Hyperglycemic rats administered tonaboxa daily for 14 days showed reduced inflammation, as evidenced by a reduction in markers for all three substances. Figure 10E -F). Quantitative results show Figure 10G In the study, compared with the undamaged control retina, all three markers GFAP, connexin 43 and Iba-1 were significantly higher in untreated hyperglycemic rats (p < 0.001), and all three treatment groups recovered to normal at 8 weeks of age, showing significantly lower levels of markers than the untreated rat retina (p < 0.001).

[0281] ***

[0282] The invention described and claimed herein has numerous attributes and embodiments, including, but not limited to, those set forth, described, or mentioned in this detailed disclosure. It is not intended to be all-encompassing, and the invention described and claimed herein is not limited to or restricted by the features or embodiments identified in this detailed disclosure, but is included for illustrative purposes only and not for limitation. Those skilled in the art will readily recognize that many components and parameters can be altered, modified, or substituted to some extent for known equivalents without departing from the scope of the invention. It should be understood that such modifications and equivalents are incorporated herein as if separately described. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, as well as any combination and all combinations of any two or more of said steps or features.

[0283] All patents, publications, scientific articles, websites, and other documents and materials referenced or mentioned herein represent the level of skill of a person skilled in the art to which this invention pertains, and each such referenced document and material is hereby incorporated by reference as if it had been individually incorporated in its entirety by reference or as set forth herein in its entirety. The applicant reserves the right to physically incorporate any and all information from any such patents, publications, scientific articles, websites, electronic information, and other references or documents into this specification. References to any applications, patents, and publications in this specification are not and should not be construed as an admission or any form of implication that they constitute valid prior art or form part of the general knowledge in any country of the world.

[0284] The specific methods and compositions described herein are representative of preferred embodiments and are exemplary, not intended to limit the scope of the invention. Other objects, aspects, and embodiments will arise in the mind of those skilled in the art in consideration of this specification, and all are included within the spirit and essence of the invention as defined by the claims. Those skilled in the art should understand that various substitutions and modifications can be made to the invention without departing from its scope and spirit. The invention suitably described herein can be practiced in the absence of any factors or limitations not specifically disclosed herein as elements. Therefore, for example, in each instance herein, and in embodiments or examples of the invention, any one of the terms “comprising,” “substantially consisting of,” and “consisting of” can be replaced in the specification with any of the other two terms. The methods and processes exemplarily described herein may be implemented in different sequences of steps, and they are not necessarily limited to the sequence of steps indicated herein or in the claims. Furthermore, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural forms unless the context clearly indicates otherwise. In no event should this patent be construed as limited to the specific embodiments, implementations, or methods specifically disclosed herein. Under no circumstances should this patent be construed as being limited by any statement made by any examiner or any other officer or employee of the Patent and Trademark Office, unless such statement is expressly and unrestrictedly or without reservation accepted in the applicant's written response. Furthermore, headings, subheadings, etc., are provided to enhance the reader's understanding of this document and should not be construed as limiting the scope of the invention. Any embodiments of aspects, implementations, or components of the invention mentioned herein are considered non-limiting.

[0285] The terms and expressions used herein are for illustrative purposes only and are not restrictive. Their use is not intended to exclude any equivalents or portions thereof of the shown and described features, but it should be recognized that various modifications are possible within the scope of the claimed invention. Therefore, it should be understood that while preferred embodiments and optional features have been specifically disclosed in the invention, modifications and variations can be made to the concepts disclosed herein by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined in the appended claims.

[0286] The invention has been described generally and in a general manner herein. Each narrower class and subgroup falling within the general scope of disclosure also forms part of the invention. This includes any incidental or negative limitation in the general specification of the invention that excludes any subject matter from the class, regardless of whether the excluded material is expressly referred to herein.

[0287] Other embodiments are within the scope of the following claims. Furthermore, in describing the features and aspects of the invention by way of the Markush group, those skilled in the art will recognize that the invention is therefore also described by any single member or subgroup of the Markush group.

Claims

1. Use of a connexin hemichannel blocker in the manufacture of a medicament for (a) restoring retinal function, (b) restoring retinal structure, (c) restoring choroidal function, and / or (d) restoring choroidal structure in a subject having a retinal disorder, wherein the connexin hemichannel blocker is N-[(3S,4S)-6-acetyl-3-hydroxy-2,2- dimethyl-3,4-dihydrochromen-4-yl]-3-chloro-4-fluorobenzamide (trabersat).

2. The use of claim 1, wherein the subject has a chronic retinal disorder.

3. The use of any one of claims 1-2, wherein the medicament is formulated for oral administration.

4. The use of any one of claims 1-2, wherein the medicament comprises an amount of connexin hemichannel blocker ranging from 10 to 400 mg.

5. The use of any one of claims 1-2, wherein the medicament comprises an amount of connexin hemichannel blocker ranging from 40 to 200 mg.

6. The use of any one of claims 1-2, wherein the medicament is formulated for administration once a day or more than once a day.

7. The use of claim 1, wherein the medicament does not comprise microparticles.

8. The use of claim 1, wherein the retinal function is selected from the group consisting of: mixed a-wave function, mixed b-wave function, electroretinogram function, and / or PII and PIII rod and cone function.

9. The use of claim 1, wherein the medicament is manufactured for restoring retinal pigment epithelium, improving retinal structural integrity, improving choroidal structural integrity, restoring retinal vascular endothelium, and / or restoring normal retinal layer structure in a subject having a retinal disorder.

10. The use of claim 1, wherein the medicament is manufactured for reducing or eliminating microaneurysms and / or macroaneurysms in a subject having a retinal disorder.

11. The use of claim 1, wherein the medicament is manufactured for improving choroidal structural integrity, restoring choroidal thickness, restoring choroidal vascular bed, improving choroidal blood flow, and / or improving choroidal vascular blood flow to the outer retina in a subject having a retinal disorder.

12. Use of a survival-promoting amount of N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl- 3,4-dihydrochromen-4-yl]-3-chloro-4-fluorobenzamide (trabersat) in the manufacture of a medicament for increasing retinal function survival in a subject in need thereof.

13. The use of claim 12, wherein the medicament is for improving photoreceptor function and / or ERG function.

14. The use of claim 12, wherein the retinal structure comprises retinal pigment epithelium, retinal vascular endothelium, and / or retinal layer structure.

15. The use of claim 12, wherein the survival-promoting amount is 10 to 200 mg per day.

16. The use of claim 12, wherein the survival-promoting amount is 20 to 100 mg per day.

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